Inhibitors of creatine transport and uses thereof

ABSTRACT

This invention relates to compounds that inhibit creatine transport and/or creatine kinase, pharmaceutical compositions including such compounds, and methods of utilizing such compounds and compositions for the treatment of cancer.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Creatine is synthesized in the liver and kidney and transportedthroughout the body to tissues with high energy demands through anactive transport system. Creatine is used by the body during times ofincreased energy demands to rapidly resynthesize ATP from ADP throughthe anaerobic conversion of phosphorylated creatine (phosphocreatine) tocreatine in a reversible reaction by the enzyme creatine kinase. Intimes of low energy demands, excess ATP can be utilized to convertcreatine to phosphocreatine. Increased expression of creatine kinasepromotes metastasis by enhancing the survival of disseminated cancercells in the liver where they encounter hepatic hypoxia. Increasedexpression of creatine kinase results in production of excessphosphocreatine which may be used as an energetic store for generatingATP needed to endure hepatic hypoxia. Inhibition of the phosphocreatinesystem through inhibition of creatine uptake and/or creatine kinase incancer cells is thus a therapeutic target for the treatment of cancerand/or metastasis.

SUMMARY OF THE INVENTION

This invention features compounds that inhibit creatine transport and/orcreatine kinase, pharmaceutical compositions including the compounds ofthe invention, and methods of utilizing those compositions forinhibition of creatine transport and/or creatine kinase (e.g., for thetreatment of cancer). Accordingly, in a first aspect the inventionfeatures a compound having the structure of Formula I:

wherein X¹ is absent, NH, or CH₂;

R¹ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R², R³, and R⁴ are independently hydrogen or optionally substitutedC₁-C₆ alkyl; and

R⁵ and R⁶ are hydrogen or NH₂;

wherein if R⁵ and R⁶ are both hydrogen or R⁵ is NH₂ and R⁶ is hydrogenthen R² is optionally substituted C₁-C₆ alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹ is hydrogen. In other embodiments, R³ and R⁴ arehydrogen. In certain embodiments, R² is hydrogen or optionallysubstituted C₁-C₆ alkyl (e.g., methyl, ethyl, isopropyl, propyl,isobutyl, or optionally substituted C₁-C₆ haloalkyl such as,trifluoromethyl.

In some embodiments, R⁵ and R⁶ are both hydrogen and R² is optionallysubstituted C₁-C₆ alkyl (e.g., methyl, ethyl, isopropyl, or isobutyl).

In other embodiments, R⁵ and R⁶ are both NH₂. In certain embodiments, R²is hydrogen. In some embodiments, R² is optionally substituted C₁-C₆alkyl (e.g., methyl or isopropyl).

In other embodiments, R⁵ is NH₂, R⁶ is hydrogen, and R² is optionallysubstituted C₁-C₆ alkyl (e.g., methyl or isopropyl).

In certain embodiments, R⁵ is hydrogen and R⁶ is NH₂. In someembodiments, R² is hydrogen. In other embodiments, R² is optionallysubstituted C₁-C₆ alkyl (e.g., methyl or isopropyl).

In certain embodiments, X¹ is absent. In some embodiments, X¹ is CH₂. Inother embodiments, X¹ is NH₂.

In certain embodiments, the compound is a compound of Table 1 (e.g.,compound 225, 229, 230, 234, or 235).

TABLE 1 Selected Phosphocreatine System Inhibitors Com- pound NumberStructure Compound Name 225

2-(2-iminoimidazolidin-1- yl)propanoic acid 226

2-(2-iminoimidazolidin-1- yl)butanoic acid 227

2-(2-iminoimidazolidin-1-yl)- 3-methylbutanoic acid 228

2-(2-iminoimidazolidin-1-yl)- 3-methylpentanoic acid 229

2-[3-amino-2- hydrazinylideneimidazolidin- 1-yl]acetic acid 230

2-[3-amino-2- hydrazinylideneimidazolidin- 1-yl]propanoic acid 231

2-[3-amino-2- hydrazinylidene- imidazolidin-1-yl]- 3-methylbutanoic acid232

2-(3-amino-2-imino- imidazolidin-1- yl)propanoic acid 233

2-(3-amino-2-imino- imidazolidin-1- yl)-3-methylbutanoic acid 234

2-[2-hydrazinylidene- imidazolidin- 1-yl]acetic acid 235

2-[2-hydrazinylidene- imidazolidin- 1-yl]propanoic acid 236

2-[2-hydrazinylidene- imidazolidin- 1-yl]-3-methylbutanoic acid

In some embodiments, the compound is a compound of Table 2 (e.g.,compound 237, 238, 241, 242, 244, 245, 247, or 248).

TABLE 2 Selected Phosphocreatine System Inhibitors Com- pound NumberStructure Compound Name 237

2-(2-imino-1,3-diazinan-1- yl)propanoic acid 238

2-(2-imino-1,3-diazinan-1- yl)butanoic acid 239

2-(2-imino-1,3-diazinan-1- yl)-3-methylbutanoic acid 240

2-(2-imino-1,3-diazinan-1- yl)-3-methylpentanoic acid 241

2-[3-amino-2- hydrazinylidene- 1,3-diazinan-1-yl]acetic acid 242

2-[3-amino-2- hydrazinylidene- 1,3-diazinan-1-yl] propionic acid 243

2-[3-amino-2- hydrazinylidene- 1,3-diazinan-1-yl]-3- methylbutanoic acid244

2-(3-amino-2-imino-1,3- diazinan-1-yl)acetic acid 245

2-(3-amino-2-imino-1,3- diazinan-1-yl)propanoic acid 246

2-(3-amino-2-imino-1,3- diazinan-1-yl)-3- methylbutanoic acid 247

2-[2-hydrazinylidene-1,3- diazinan-1-yl]acetic acid 248

2-[2-hydrazinylidene-1,3- diazinan-1-yl]propanoic acid 249

2-[2-hydrazinylidene-1,3- diazinan-1-yl]-3- methylbutanoic acid

In other embodiments, the compound is a compound of Table 3 (e.g.,compound 250 or 251).

TABLE 3 Selected Phosphocreatine System Inhibitors Com- pound NumberStructure Compound Name 250

2-(3-imino-1,2,4-triazinan-2- yl)acetic acid 251

2-(3-imino-1,2,4-triazinan-2- yl)propanoic acid 252

2-(3-imino-1,2,4-triazinan-2-yl)-3- methylbutanoic acid

In another aspect, the invention features a compound having thestructure of Formula II:

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl;

X² is S or NR¹²;

m is 0 or 1;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁸ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, NH₂,optionally substituted C₁-C₆ alkyl, or R⁸ or R⁹ can combine with R¹⁰ orR¹¹ to form an optionally substituted C₃-C₆ cycloalkyl ring or with R¹²to form an optionally substituted C₃-C₆ heterocycle;

R¹⁰ and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₆ alkyl, or R¹⁰ or R¹¹ can combine with R⁸ or R⁹ to forman optionally substituted C₃-C₆ cycloalkyl ring;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² can combinewith R⁸ or R⁹ to form an optionally substituted C₃-C₆ heterocycle, and

wherein if R⁹ is halo then R⁸ is halo or optionally substituted C₁-C₆alkyl,

or a pharmaceutically acceptable salt thereof.

In some embodiments, if Q¹ is optionally substituted 2-pyridyl then R¹²is hydrogen,

In some embodiments, R⁷ is hydrogen. In other embodiments, m is 1. Incertain embodiments, R⁹ is hydrogen, deuterium, or halo (e.g., fluoro).In some embodiments, R¹¹ is hydrogen or deuterium.

In other embodiments, R⁸ and R¹⁰ combine to form an optionallysubstituted C₃-C₆ cycloalkyl ring (e.g., cyclopropyl or cyclobutyl). Incertain embodiments, R¹⁰ and R¹¹ are deuterium. In other embodiments, R⁸and R⁹ are deuterium. In some embodiments, both R⁸ and R⁹ are halo(e.g., fluoro).

In other embodiments, R¹⁰ is optionally substituted C₁-C₆ alkyl (e.g.,optionally substituted C₁-C₆ haloalkyl such as, trifluoromethyl).

In certain embodiments, R⁸ is NH₂. In some embodiments, R¹⁰ isoptionally substituted C₁-C₆ alkyl (e.g., methyl).

In other embodiments, Q¹ is optionally substituted amidino (e.g.,

In certain embodiments, X² is NR¹². In some embodiments, R⁸ and R¹²combine to form an optionally substituted C₃-C₆ heterocycle (e.g.,azetidine). In other embodiments, R¹² is hydrogen. In certainembodiments, X² is S.

In some embodiments, Q¹ is optionally substituted 2-pyridyl (e.g.,

In other embodiments, X² is NR¹² and R¹² is hydrogen.

In some embodiments, the compound has the structure of Formula II:

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl;

X² is S or NR¹²;

m is 1 or 2;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁸ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, NH₂,optionally substituted C₁-C₃ alkyl, or R⁸ and R⁹ combine with the atomsto which they are attached to form an optionally substituted

C₃-C₆ cycloalkyl ring; or R⁸ or R⁹ combine with R¹⁰ or R¹¹ with theatoms to which they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R⁸ or R⁹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₅ heterocycle;

R¹⁰ and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₄ alkyl, optionally substituted C₂-C₆ alkenyl,optionally substituted C₂-C₆ alkynyl or R¹⁰ and R¹¹ combine with theatoms to which they are attached to form an optionally substituted C₃-C₆cycloalkyl ring; or R¹⁰ or R¹¹ combine with R⁸ or R⁹ with the atoms towhich they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R¹⁰ or R¹¹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₅ heterocycle;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² combineswith R⁸, R⁹, R¹⁰, or R¹¹ with the atoms to which they are attached toform an optionally substituted C₃-C₅ heterocycle,

wherein if Q¹ is optionally substituted 2-pyridyl and R⁸ is optionallysubstituted C₁-C₃ alkyl, halo, or hydroxyl then at least one of R¹⁰ andR¹¹ are independently deuterium, optionally substituted C₂-C₆ alkenyl,optionally substituted C₂-C₆ alkynyl or R¹⁰ and R¹¹ combine with theatoms to which they are attached to form an optionally substituted C₃-C₆cycloalkyl ring; or R¹⁰ or R¹¹ combine with R⁹ with the atoms to whichthey are attached to form an optionally substituted C₃-C₄ cycloalkylring; or R¹⁰ or R¹¹ combine with R¹² with the atoms to which they areattached to form an optionally substituted C₃-C₅ heterocycle;

wherein if Q¹ is optionally substituted 2-pyridyl and R¹⁰ is optionallysubstituted C₁-C₄ alkyl then at least one of R⁸ and R⁹ are independentlydeuterium, hydroxyl, NH₂, optionally substituted C₁-C₃ alkyl, or R⁸ andR⁹ combine with the atoms to which they are attached to form anoptionally substituted C₃-C₆ cycloalkyl ring; or R⁸ or R⁹ combine withR¹¹ with the atoms to which they are attached to form an optionallysubstituted C₃-C₄ cycloalkyl ring; or R⁸ or R⁹ combine with R¹² with theatoms to which they are attached to form an optionally substituted C₃-C₅heterocycle;

wherein if m is 1 and R⁸ is hydrogen, fluoro, hydroxyl, or methyl thenat least one of R⁹, R¹⁰, and R¹¹ is not hydrogen;

wherein if m is 1 and R¹⁰ is methyl then at least one of R⁸, R⁹, and R¹¹is not hydrogen;

wherein if m is 1 and R⁸ is NH₂ and R¹⁰ is hydrogen, methyl, or—CH₂CH₂OH then at least one of R⁹ or R¹¹ is not hydrogen;

or a pharmaceutically acceptable salt thereof.

In some embodiments, if Q¹ is optionally substituted 2-pyridyl then R¹²is hydrogen,

In another aspect, the invention features a compound having thestructure of Formula V:

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl;

m is 1 or 2;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁸ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, NH₂,optionally substituted C₁-C₃ alkyl, or R⁸ and R⁹ combine with the atomsto which they are attached to form an optionally substituted C₃-C₆cycloalkyl ring; or R⁸ or R⁹ combine with R¹⁰ or R¹¹ with the atoms towhich they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R⁸ or R⁹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₅ heterocycle;

R¹⁰ and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₄ alkyl, optionally substituted C₂-C₆ alkenyl,optionally substituted C₂-C₆ alkynyl or R¹⁰ and R¹¹ combine with theatoms to which they are attached to form an optionally substituted C₃-C₆cycloalkyl ring; or R¹⁰ or R¹¹ combine with R⁸ or R⁹ with the atoms towhich they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R¹⁰ or R¹¹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₄ heterocycle;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² combineswith R⁸ or R⁹ with the atoms to which they are attached to form anoptionally substituted C₃-C₅ heterocycle, or R¹² combines with R¹⁰ orR¹¹ with the atoms to which they are attached to form an optionallysubstituted C₃-C₄ heterocycle

wherein if m is 1 and R⁸ is hydrogen, halo, hydroxyl, or methyl then atleast one of R⁹, R¹⁰, and R¹¹ is not hydrogen;

wherein if m is 1 and R¹⁰ is methyl then at least one of R⁸, R⁹, and R¹¹is not hydrogen;

wherein if m is 1 and R⁸ is NH₂ and R¹⁰ is hydrogen, methyl, or—CH₂CH₂OH then at least one of R⁹ or R¹¹ is not hydrogen;

wherein if m is 1, R⁸ is halo, and R¹⁰ is optionally substituted C₁-C₄alkyl then at least one of R⁹ and R¹⁰ is not hydrogen;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R⁷ is hydrogen. In other embodiments Q¹ isoptionally substituted amidino (e.g., Q¹ is

In some embodiments, Q¹ is optionally substituted 2-pyridyl (e.g.,2-pyridyl).

In some embodiments, R⁸ combines with R¹⁰ with the atoms to which theyare attached to form an optionally substituted C₃-C₄ cycloalkyl ring. Insome embodiments, the compound has the structure of Formula VI:

wherein a is 0 or 1.

In other embodiments, the compound has the structure of Formula VII:

In some embodiments, R⁹ is hydrogen, hydroxyl, or NH₂. In otherembodiments, R¹¹ is hydrogen.

In some embodiments, m is 1 and R¹⁰ is deuterium, optionally substitutedC₁-C₄ alkyl, optionally substituted C₂-C₆ alkenyl, optionallysubstituted C₂-C₆ alkynyl, or R¹⁰ and R¹¹ combine with the atoms towhich they are attached to form an optionally substituted C₃-C₆cycloalkyl ring.

In other embodiments, R¹⁰ is deuterium. In some embodiments, R¹¹ isdeuterium. In other embodiments, R⁸ and R⁹ are both deuterium. In someembodiments, R¹⁰ is optionally substituted C₁-C₄ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl. Inother embodiments, R¹⁰ is methyl, ethyl, n-propyl, iso-propyl, —CD₃,—CF₃, —CH₂F, —CHF₂, —CH═CH₂, or —C≡CH. In some embodiments, R¹¹ ishydrogen or methyl. In other embodiments, R⁸ is hydrogen. In someembodiments, R⁹ is hydrogen, NH₂, or methyl. In other embodiments, R¹⁰and R¹¹ combine with the atoms to which they are attached to form anoptionally substituted C₃-C₆ cycloalkyl ring (e.g., an optionallysubstituted C₃-C₄ cycloalkyl ring such as cyclopropyl or cyclobutyl). Insome embodiments, R⁸ and R⁹ are both hydrogen.

In other embodiments, R⁸ is halo, optionally substituted C₁-C₃ alkyl, orR⁸ and R⁹ combine with the atoms to which they are attached to form anoptionally substituted C₃-C₆ cycloalkyl ring. In some embodiments, R⁸ ishalo (e.g., fluoro). In other embodiments, R⁹ is halo (e.g., fluoro). Insome embodiments, R⁸ is optionally substituted C₁-C₃ alkyl (e.g.,methyl). In other embodiments, R⁹ is optionally substituted C₁-C₃ alkyl(e.g., methyl). In some embodiments, R⁸ and R⁹ combine with the atoms towhich they are attached to form an optionally substituted C₃-C₆cycloalkyl ring (e.g., an optionally substituted C₃-C₄ cycloalkyl ringsuch as cyclopropyl or cyclobutyl). In some embodiments, R¹⁰ and R¹¹ areboth hydrogen.

In certain embodiments of the compounds of Formula V, R¹² is hydrogen.

In some embodiments, R¹² combines with R⁸ with the atoms to which theyare attached to form an optionally substituted C₃-C₅ heterocycle (e.g.,an optionally substituted C₄-C₅ heterocycle).

In some embodiments, the compound has the structure of Formula VIII:

wherein b and c are each, independently, 0 or 1.

In some embodiments, R¹⁹ is hydrogen or optionally substituted C₁-C₄alkyl (e.g., methyl). In other embodiments, R¹¹ is hydrogen. In someembodiments, R⁹ is hydrogen, halo (e.g., fluoro), hydroxyl, NH₂,optionally substituted C₁-C₃ alkyl (e.g., methyl).

In other embodiments, R¹² combines with R¹⁹ with the atoms to which theyare attached to form an optionally substituted C₃-C₄ heterocycle.

In some embodiments, the compound has the structure of Formula IX:

In some embodiments, R¹¹ is hydrogen. In other embodiments, R⁸ and R⁹are both hydrogen.

In certain embodiments of any of the compounds of Formula V, Q¹ is

In some embodiments, the compound of Formula II or Formula V is any oneof compounds 253-262 or 327-385 in Table 4.

In some embodiments, the compound of Formula II or Formula V is any oneof compounds 263-274 or 386-436 in Table 5.

In another aspect the invention features a compound selected from anyone of compounds 253-262 and 327-385 in Table 4. In some embodiments,the compound is any one of compounds 258, 327-338, 340, 343-348,351-352, 366-367, 369-370, 372-375, and 379-385 in Table 4.

TABLE 4 Selected Phosphocreatine System Inhibitors Compound NumberStructure Compound Name 253

cis-2- carbamimidamidocyclopropane- 1-carboxylic acid 254

trans-2- carbamimidamidocyclopropane- 1-carboxylic acid 255

3-carbamimidamido(²H)propanoic acid 256

3-carbamimidamido(3,3- ²H₂)propanoic acid 257

3-carbamimidamido-2,2- difluoropropanoic acid 258

1-carbamimidoylazetidine-3- carboxylic acid 259

cis-2- carbamimidamidocyclobutane-1- carboxylic acid 260

trans-2- carbamimidamidocyclobutane-1- carboxylic acid 261

3-guanidino-4,4,4- trifluorobutanoic acid 262

2-amino-3-guanidino-4,4,4- trifluorobutanoic acid 327

(1R,2S)-2- carbamimidamidocyclopropane- 1-carboxylic acid 328

(1S,2R)-2- carbamimidamidocyclopropane- 1-carboxylic acid 329

(1R,2R)-2- carbamimidamidocyclopropane- 1-carboxylic acid 330

(1S,2S)-2- carbamimidamidocyclopropane- 1-carboxylic acid 331

(1R,2S)-2- carbamimidamidocyclobutane-1- carboxylic acid 332

(1S,2R)-2- carbamimidamidocyclobutane-1- carboxylic acid 333

(1R,2R)-2- carbamimidamidocyclobutane-1- carboxylic acid 334

(1S,2S)-2- carbamimidamidocyclobutane-1- carboxylic acid 335

(2R,3R)-1-carbamimidoyl-2- methylazetidine-3-carboxylic acid 336

(2R,3S)-1-carbamimidoyl-2- methylazetidine-3-carboxylic acid 337

(2S,3R)-1-carbamimidoyl-2- methylazetidine-3-carboxylic acid 338

(2S,3S)-1-carbamimidoyl-2- methylazetidine-3-carboxylic acid 339

1-carbamimidoyl-3- hydroxyazeitidine-3-carboxylic acid 340

3-amino-1- carbamimidoylazetidine-3- carboxylic acid 341

1-carbamimidoyl-3- fluoroazetidine-3-carboxylic acid 342

1-carbamimidoyl-3- methylazetidine-3-carboxylic acid 343

(S)-3-guanidino-4,4,4- trifluorobutanoic acid 344

(R)-3-guanidino-4,4,4- trifluorobutanoic acid 345

(2S,3S)-2-amino-3- carbamimidamido-4,4,4- trifluorobutanoic acid 346

(2S,3R)-2-amino-3- carbamimidamido-4,4,4- trifluorobutanoic acid 347

(2R,3R)-2-amino-3- carbamimidamido-4,4,4- trifluorobutanoic acid 348

(2R,3S)-2-amino-3- carbamimidamido-4,4,4- trifluorobutanoic acid 349

(3R)-3-carbamimidamido(4,4,4- ²H₃)butanoic acid 350

(3S)-3-carbamimidamido(4,4,4- ²H₃)butanoic acid 351

(3S)-3-carbamimidamido-4,4- difluorobutanoic acid 352

(3R)-3-carbamimidamido-4,4- difluorobutanoic acid 353

3-carbamimidamido-4- fluorobutanoic acid 354

(3S)-3-carbamimidamidopent-4- enoic acid 355

(3R)-3-carbamimidamidopent-4- enoic acid 356

(3S)-3-carbamimidamidopent-4- ynoic acid 357

(3R)-3-carbamimidamidopent-4- ynoic acid 358

3-carbamimidamidopentanoic acid 359

(3R)-3- carbamimidamidopentanoic acid 360

(3S)-3- carbamimidamidopentanoic acid 361

(3R)-3- carbamimidamidohexanoic acid 362

(3S)-3- carbamimidamidohexanoic acid 363

3-carbamimidamido-4- methylpentanoic acid 364

(3S)-3-carbamimidamido-4- methylpentanoic acid 365

(3R)-3-carbamimidamido-4- methylpentanoic acid 366

3-carbamimidamido-2,2- dimethypropanoic acid 367

1- (carbamimidamidomethyl) cyclopropane-1-carboxylic acid 368

1- (carbamimidamidomethyl) cyclobutane-1-carboxylic acid 369

3-carbamimidamido-3- methylbutanoic acid 370

2-(1- carbamimidamidocyclopropyl) acetic acid 371

2-(1- carbamimidamidocyclobutyl)acetic acid 372

(2R,3R)-3-carbamimidamido-2- methylbutanoic acid 373

(2S,3R)-3-carbamimidamido-2- methylbutanoic acid 374

(2R,3S)-3-carbamimidamido-2- methylbutanoic acid 375

(2S,3S)-3-carbamimidamido-2- methylbutanoic acid 376

1-carbamimidoylpyrrolidine-3- carboxylic acid 377

(3S)-1-carbamimidoylpyrrolidine- 3-carboxylic acid 378

(3R)-1-carbamimidoylpyrrolidine- 3-carboxylic acid 379

2-[(2R)-1-carbamimidoylazetidin- 2-yl]acetic acid 380

2-[(2S)-1-carbamimidoylazetidin- 2-yl]acetic acid 381

cis-3- carbamimidamidocyclobutane-1- carboxylic acid 382

trans-3- carbamimidamidocyclobutane-1- carboxylic acid 383

3-carbamimidamido-1- hydroxycyclobutane-1- carboxylic acid 384

1-amino-3- carbamimidamidocyclobutane-1- carboxylic acid 385

2-(1-carbamimidoylazetidin-3- yl)acetic acid

In another aspect, the invention features a compound selected from anyone of compounds 263-274 and 386-436 in Table 5.

TABLE 5 Selected Phosphocreatine System Inhibitors Com- pound Num- berStructure Compound Name 263

(2S)-2-amino-3-[(pyridin- 2-yl)amino]propanoic acid 264

(2S,3S)-2-amino-3- [(pyridin-2- yl)amino]butanoic acid 265

(2S,3R)-2-amino-3- [(pyridin-2- yl)amino]butanoic acid 266

cis-2-[(pyridin-2-yl) amino]cyclopropane-1- carboxylic acid 267

trans-2-[(pyridin-2- yl)amino]cyclopropane-1- carboxylic acid 268

3-[(pyridin-2- yl)amino](²H₄)propanoic acid 269

3-[(pyridin-2- yl)amino](3,3- ²H₂)propanoic acid 270

2,2-difluoro-3-[(pyridin- 2-yl)amino]propanoic acid 271

cis-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 272

trans-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 273

4,4,4-trifluoro-3- [(pyridine-2- yl)amino]butanoic acid 274

2-amino-4,4,4-trifluoro-3- [(pyridine-2- yl)amino]butanoic acid 386

(2R)-2-amino-3-[(pyridin- 2-yl)amino]propanoic acid 387

(2R,3S)-2-amino-3- [(pyridin-2- yl)amino]butanoic acid 388

(2R,3R)-2-amino-3- [(pyridin-2- yl)amino]butanoic acid 389

(1R,2S)-2-[(pyridin-2- yl)amino]cyclopropane-1- carboxylic acid 390

(1S,2R)-2-[(pyridin-2- yl)amino]cyclopropane-1- carboxylic acid 391

(1R,2R)-2-[(pyridin-2- yl)amino]cyclopropane-1- carboxylic acid 392

(1S,2S)-2-[(pyridin-2- yl)amino]cyclopropane-1- carboxylic acid 393

(1R,2S)-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 394

(1S,2R)-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 395

(1R,2R)-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 396

(1S,2S)-2-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 397

(2R,3R)-2-methyl-1- (pyridin-2-yl)azetidine-3- carboxylic acid 398

(2R,3S)-2-methyl-1- (pyridin-2-yl)azetidine-3- carboxylic acid 399

(2S,3R)-2-methyl-1- (pyridin-2-yl)azetidine-3- carboxylic acid 400

(2S,3S)-2-methyl-1- (pyridin-2-yl)azetidine-3- carboxylic acid 401

3-hydroxy-1-(pyridin-2- yl)azetidine-3-carboxylic acid 402

3-amino-1-(pyridin-2- yl)azetidine-3-carboxylic acid 403

3-fluoro-1-(pyridin-2- yl)azetidine-3-carboxylic acid 404

3-methyl-1-(pyridin-2- yl)azetidine-3-carboxylic acid 405

(3S)-4,4,4-trifluoro-3- [(pyridin-2- yl)amino]butanoic acid 406

(3R)-4,4,4-trifluoro-3- [(pyridin-2- yl)amino]butanoic acid 407

(2S,3S)-2-amino-4,4,4- trifluoro-3-[(pyridin-2- yl)amino]butanoic acid408

(2S,3R)-2-amino-4,4,4- trifluoro-3-[(pyridin-2- yl)amino]butanoic acid409

(2R,3R)-2-amino-4,4,4- trifluoro-3-[(pyridin-2- yl)amino]butanoic acid410

(2R,3S)-2-amino-4,4,4- trifluoro-3-[(pyridin-2- yl)amino]butanoic acid411

(3R)-3-[(pyridin-2- yl)amino](4,4,4- ²H₃)butanoic acid 412

(3S)-3-[(pyridin-2- yl)amino](4,4,4- ²H₃)butanoic acid 413

(3S)-4,4-difluoro-3- [(pyridin-2- yl)amino]butanoic acid 414

(3R)-4,4-difluoro-3- [(pyridin-2- yl)amino]butanoic acid 415

4-fluoro-3-[(pyridin-2- yl)amino]butanoic acid 416

(3S)-3-[(pyridin-2- yl)amino]pent-4-enoic acid 417

(3R)-3-[(pyridin-2- yl)amino]pent-4-enoic acid 418

(3S)-3-[(pyridin-2- yl)amino]pent-4-ynoic acid 419

(3R)-3-[(pyridin-2- yl)amino]pent-4-ynoic acid 420

(3R)-3-[(pyridin-2- yl)amino]pentanoic acid 421

(3S)-3-[(pyridin-2- yl)amino]pentanoic acid 422

2,2-dimethyl-3-[(pyridin- 2-yl)amino]propanoic acid 423

1-{[(pyridin-2- yl)amino]methyl} cyclopropane-1- carboxylic acid 424

1-{[(pyridin-2- yl)amino]methyl} cyclobutane-1-carboxylic acid 425

3-methyl-3-[(pyridin-2- yl)amino]butanoic acid 426

2-{-1-[(pyridin-2- yl)amino]cyclopropyl} acetic acid 427

2-{-1-[(pyridin-2- yl)amino]cyclobutyl} acetic acid 428

(2R,3R)-2-methyl-3- [(pyridin-2- yl)amino]butanoic acid 429

(2S,3R)-2-methyl-3- [(pyridin-2- yl)amino]butanoic acid 430

(2R,3S)-2-methyl-3- [(pyridin-2- yl)amino]butanoic acid 431

(2S,3S)-2-methyl-3- [(pyridin-2- yl)amino]butanoic acid 432

2-[(2R)-1-(pyridin-2- yl)azetidin-2-yl]acetic acid 433

2-[(2S)-1-(pyridin-2- yl)azetidin-2-yl]acetic acid 434

cis-3-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 435

trans-3-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid 436

1-hydroxy-3-[(pyridin-2- yl)amino]cyclobutane-1- carboxylic acid

In another aspect, the invention features a compound selected from anyone of compounds 275-286 in Table 6.

TABLE 6 Selected Phosphocreatine System Inhibitors Compound NumberStructure Compound Name 275

(2R)-2-amino-3- (carbamimidoylsulfanyl)propanoic acid 276

(2S,3S)-2-amino-3- (carbamimidoylsulfanyl)butanoic acid 277

(2S,3R)-2-amino-3- (carbamimidoylsulfanyl)butanoic acid 278

cis-2- (carbamimidoylsulfanyl)cyclopropane-1- carboxylic acid 279

trans-2- (carbamimidoylsulfanyl)cyclopropane-1- carboxylic acid 280

3-(carbamimidoylsulfanyl)(2,2- ²H₄)propanoic acid 281

3-(carbamimidoylsulfanyl)(2,2- ²H)propanoic acid 282

3-(carbamimidoylsulfanyl)-2,2- difluoropropanoic acid 283

cis-2-(carbamimidoylsulfanyl)cyclobutane- 1-carboxylic acid 284

trans-2- (carbamimidoylsulfanyl)cyclobutane-1- carboxylic acid 285

3-(carbamimidoylsulfanyl)-4,4,4- trifluorobutanoic acid 286

2-amino-3-(carbamimidoylsulfanyl)-4,4,4- trifluorobutanoic acid

In another aspect, the invention features a compound of Table 7 that issubstantially enantiomerically pure.

TABLE 7 Selected Phosphocreatine System Inhibitors Com- pound Num- berStructure Compound Name 219

(3R)-3- carbamimidamidobutanoic acid 220

(3S)-3- carbamimidamidobutanoic acid 221

(2S,3R)-2-amino-3- carbamimidamidobutanoic acid 222

(2S,3S)-2-amino-3- carbamimidamidobutanoic acid 437

(2R,3R)-2-amino-3- carbamimidamidobutanoic acid 438

(2R,3S)-2-amino-3- carbamimidamidobutanoic acid 439

(2S)-1- carbamimidoylazetidine- 2-carboxylic acid 440

(2R)-1- carbamimidoylazetidine- 2-carboxylic acid 223

(3R)-3-[(pyridin-2- yl)amino]butanoic acid 224

(3S)-3-[(pyridin-2- yl)amino]butanoic acid 441

(3R)-3-[(pyridin-2- yl)amino]hexanoic acid 442

(3S)-4-methyl-3-[(pyridin- 2-yl)amino]pentanoic acid 443

(3S)-4-methyl-3-[(pyridin- 2-yl)amino]pentanoic acid 444

(3R)-4-methyl-3- [(pyridin-2- yl)amino]pentanoic acid 445

(3S)-1-(pyridin-2- yl)pyrrolidine-3- carboxylic acid 446

(3R)-1-(pyridin-2- yl)pyrrolidine-3- carboxylic acid

In another aspect, the invention features a compound having thestructure:A-B  Formula III

wherein A is a inhibitor of creatine transport and/or creatine kinasecomprising an amidino group;

B has the structure:

wherein n is 0 or 1;

Q² is hydroxyl, optionally substituted amino, or —SO₂OH; and

R¹³ and R¹⁴ are independently hydrogen, —CO₂H, or combine to form C═O;

wherein B is conjugated to A at one of the amidino nitrogens,

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹⁴ is hydrogen. In other embodiments, R¹³ is—CO₂H. In certain embodiments, R¹³ is hydrogen. In some embodiments, R¹³and R¹⁴ combine to form C═O. In other embodiments, n is 0. In certainembodiments, n is 1. In some embodiments, Q² is optionally substitutedamino (e.g., —NH₂ or

In other embodiments, Q² is hydroxyl. In certain embodiments, Q² is—SO₂OH.

In some embodiments, B has the structure:

In other embodiments, A has the structure of any of the foregoingcompounds.

In another aspect, the invention features a compound selected from anyone of compounds 1-218 or 323-326 in Table 8.

TABLE 8 Selected Phosphocreatine System Inhibitors Com- pound Num- berStructure Compound Name  1

(1-methylguanidinomethyl)phosphinic acid  2

[(2-iminoimidazolidin-1-yl)methyl]phosphinic acid  3

[(2-imino-3-(phosphono)imidazolidin-1- yl)methyl]phosphinic acid  4

{1-methyl-3-(phosphono)guanidinomethyl}phosphinic acid  5

1-(N-phosphonocarbamimidoyl)azetidine-2-carboxylic acid  6

1-(N-phosphonocarbamimidoyl)pyrrolidine-2-carboxylic acid  7

1-carbamimidoylazetidine-2-carboxylic acid  8

1-carbamimidoylpyrrolidine-2-carboxylic acid  9

2-(1-ethylguanidino)acetic acid  10

2-(1-methylguanidino)acetic acid (Creatine)  11

2-(1-methylguanidino)propanoic acid  12

2-(3-phosphono-1-propylguanidino)acetic acid  13

2-(1-phosphonocarbamimidamido)acetic acid  14

2-(1-propylguanidino)acetic acid  15

2-(2-amino-1H-imidazol-1-yl)acetic acid  16

2-(2-imino-1,3-diazinan-1-yl)acetic acid(1-Carboxymethyl-2-iminohexahydropyrimidine)  17

2-(2-imino-3-phosphonoimidazolidin-1-yl)acetic acid (N-PhosphorylCyclocreatine)  18

2-(2-iminoimidazolidin-1-yl)acetic acid (Cyclocreatine)  19

2-(3-phosphono-1-ethylguanidino)acetic acid  20

2-(3-phosphono-1-methylguanidino)acetic acid (N-Phosphoryl Creatine)  21

2-(3-phosphono-1-methylguanidino)propionic acid  22

2-carbamimidamidoacetic acid (Guanidinoacetic acid)  23

3-(1-methylguanidino)propanoic acid (Homocreatine)  24

3-(3-phosphonoguanidino)propionic acid  25

3-(2-imino-3-phosphonoimidazolidin-1-yl)propanoic acid (N-PhosphorylHomocyclocreatine)  26

3-(2-iminoimidazolidin-1-yl)propanoic acid (Homocyclocreatine)  27

3-(3-phosphono-1-methylguanidino)propionic acid (Phosohomocreatine)  28

3-carbamimidamidopropanoic acid (β-Guanidinopropionic acid; β-GPA)  29

3-carbamimidoyl-3-methylpropanoic acid (Carbocreatine)  30

2-(1,3-dimethylguanidino)acetic acid  31

2-carbamimidamidobutanedioic acid  32

2-carbamimidamidobutanoic acid  33

2-carbamimidamidopropanoic acid  34

3-carbamimidamidobutanoic acid (β-DL-Guanidinobutanoicic acid; β-GBA) 35

4-carbamimidamidobutanoic acid  36

2-({[N′-hydroxycarbamimidoyl]methyl}amino)acetic acid  37

2-({3-[(carboxymethylidene)amino]guanidino}imino)acetic acid  38

2-({bis[(2-aminoethyl)amino]methylidene}amino)acetic acid  39

2-({bis[(2-hydroxyethyl)amino]methylidene}amino)acetic acid  40

2-({bis[(3,5-dimethyl-1H-pyrazol-1- yl)amino]methylidene}amino)aceticacid  41

2-({bis[(carbamoylamino)amino]methylidene}amino)acetic acid  42

2-(1-aminoguanidino)acetic acid  43

2-(1,3-diaminoguanidino)acetic acid  44

2-{N-(2-amino)ethanimidamido}acetic acid  45

2-(carbamimidamidoamino)-2-phenylacetic acid  46

2-(carbamimidamidoamino)-3-phenylpropanoic acid  47

2-(carbamimidamidoamino)acetic acid  48

2-(carbamimidamidoamino)butanoic acid  49

2-(carbamimidamidoimino)-2-phenylacetic acid  50

2-(carbamimidamidoimino)-3-phenylpropanoic acid  51

2-(carbamimidamidoimino)acetic acid  52

2-(carbamimidamidoimino)butanoic acid  53

2-(carbamimidamidoimino)octanoic acid  54

2-(carbamimidamidoimino)propanoic acid  55

2-(N-carbamimidoylimidamido)acetic acid  56

2-[(1-methylguanidino)imino]acetic acid  57

2-[(2-amino-1H-imidazol-1-yl)amino]acetic acid  58

2-[(2,10-dimethyl-2,5,7,10-tetraazaundecan-6- ylidene)amino]acetic acid 59

2-[(3-amino-4H-1,2,4-triazol-4-yl)amino]acetic acid  60

2-[(6-oxo-1,2,4,5-tetrazinan-3-ylidene)amino]acetic acid  61

2-(biguanide)acetic acid  62

2-(biguanide)ethane-1-sulfonic acid  63

2-[(carbamimidoylmethyl)amino]acetic acid  64

2-[(di{[(methoxycarbonyl)amino]amino}methylidene) amino]acetic acid  65

2-[(di{[(acetyl)amino]amino}methylidene)amino]acetic acid  66

2-[(dihydrazinylmethylidene)amino]-2-phenylacetic acid  67

2-[(dihydrazinylmethylidene)amino]-3-methylbutanoic acid  68

2-[(dihydrazinylmethylidene)amino]-3-phenylpropanoic acid  69

2-[(dihydrazinylmethylidene)amino]acetic acid  70

2-[(dihydrazinylmethylidene)amino]propanoic acid  71

2-[[(2,2-dimethylhydrazin-1- yl)(hydrazinyl)methylidene]amino]aceticacid  72

2-[{[(carbamoylamino)amino](hydrazinyl)methylidene} amino]acetic acid 73

2-[{hydrazinyl[(1H-pyrazol-5- yl)amino]methylidene}amino]acetic acid  74

2-[{hydrazinyl[(2- hydroxyethyl)amino]methylidene}amino]acetic acid  75

2-[{hydrazinyl[(morpholin-4- yl)amino]methylidene}amino]acetic acid  76

2-[{hydrazinyl[2-(pyridin-2-yl)hydrazin-1- yl]methylidene}amino]aceticacid  77

2-[2-(2-aminoethyl)carbamimidamido]acetic acid  78

2-[2-(4,5-dihydro-1H-imidazol-2-yl)hydrazin-1- ylidene]acetic acid  79

2-[2-(pyridin-2-yl)hydrazin-1-ylidene]acetic acid  80

2-[2-aminocarbamimidamido]acetic acid  81

2[2-hydroxycarbamimidamido]acetic acid  82

2-{[2- [(phenylmethylidene)amino]carbamimidamido]amino}acetic acid  83

2-{[2-[(propan-2-yl)amino]carbamimidamido]amino}acetic acid  84

2-{[2-[(propan-2- ylidene)amino]carbamimidamido]amino}acetic acid  85

2-{[2-aminocarbamimidamido]amino}acetic acid  86

2-{[2-methylcarbamimidamido]imino}acetic acid  87

2-{[2-nitrocarbamimidamido]imino}acetic acid  88

2-{[4-amino-1,2,4-triazinan-3-ylidene]amino}acetic acid  89

2-{[6-methyl-1,2,3,4-tetrahydro-1,2,4,5-tetrazin-3- ylidene]amino}aceticacid  90

3-({[N-hydroxycarbamimidoyl]methyl}amino)propanoic acid  91

3-{N-(2-amino)ethanimidamido}propanoic acid  92

3-(carbamimidamidoamino)propanoic acid  93

3-(N-carbamimidoylimidamido)propanoic acid  94

3-(biguanide)propanoic acid  95

3-[(carbamimidoylmethyl)amino]propanoic acid  96

3-[(dihydrazinylmethylidene)amino]propanoic acid  97

3-[2-aminocarbamimidamido]propanoic acid  98

3-hydrazinylidene-1,2,4-triazinan-5-one  99

3-hydrazinylidene-2-methyl-1,2,4-triazinan-6-one 100

3-hydrazinylidene-2,3,4,5-tetrahydro-1,2,4-triazin-5-one 101

3-imino-1,2,4-triazinan-5-one 102

3-imino-2,3,4,5-tetrahydro-1,2,4-triazin-5-one 103

4-(biguanide)butanoic acid 104

4-[(dihydrazinylmethylidene)amino]butanoic acid 105

4-[2-aminocarbamimidamido]butanoic acid 107

ethyl 3-(biguanide)propanoate 108

methyl 2-(carbamimidamidoamino)acetate 109

methyl 2-(carbamimidamidoimino)acetate 110

methyl 3-(N-carbamimidoylimidamido)propanoate 111

methoxy(1-methylcarbamimidamidomethyl)phosphinic acid 112

2-(1H-imidazol-2-yl)acetic acid 113

2-[1-amino-2-methylguanidino]acetic acid 114

3-(1,4,5,6-tetrahydropyrimidin-2-yl)propanoic acid 115

3-(1H-imidazol-2-yl)propanoic acid 116

3-(4,5-dihydro-1H-imidazol-2-yl)propanoic acid 117

3-(4H-1,2,4-triazol-3-yl)propanoic acid 118

3-[(3,4-dihydro-2H-pyrrol-5-yl)amino]propanoic acid 119

(1R,2S)-2-carbamimidamidocyclohexane-1-carboxylic acid 120

(1R,2S)-2-carbamimidamidocyclopentane-1-carboxylic acid 121

(1S,2S)-2-carbamimidamidocyclohexane-1-carboxylic acid 122

(1S,2S)-2-carbamimidamidocyclopentane-1-carboxylic acid 123

(2-carbamimidamidoethyl)phosphonic acid 124

(2R)-2-amino-3-carbamimidamidopropanoic acid 125

(2S)-2-amino-3-carbamimidamidopropanoic acid 126

(2S)-2-amino-5-carbamimidamidopentanoic acid (L-Arg) 127

1-[2-(1H-1,2,3,4-tetrazol-5-yl)ethyl]guanidine 128

3-[(4,5-dihydro-1H-imidazol-2-yl)amino]propanoic acid 129

1-[2-(2-sulfanyl-1H-imidazol-5-yl)ethyl]guanidine 130

1-carbamimidoylpiperidine-3-carboxylic acid 131

2-(1-carbamimidoylpiperidin-2-yl)acetic acid 132

2-(1H-imidazol-4-yl)acetic acid 133

2-(5-carbamimidoyl-1H-pyrrol-2-yl)acetic acid 134

2-(6-aminopyridin-2-yl)acetic acid 135

2-(carbamimidamidomethyl)heptanoic acid 136

2-(carbamimidamidooxy)acetic acid 137

2-(carbamimidoylsulfanyl)acetic acid 138

2-[(carbamimidoylmethyl)sulfanyl]acetic acid 139

2-amino-1-(2-carboxylatoethyl)pyridin-1-ium 140

2-amino-3-(1H-imidazol-5-yl)propanoic acid 141

2-phenyl-3-carbamimidamidopropanoic acid 142

2-carbamimidamidoethane-1-sulfonic acid 143

3-(3-hexylguanidino)propanoic acid 144

3-(3-methylguanidino)propanoic acid 145

3-(1H-imidazol-1-yl)propanoic acid 147

3-(2-carbamimidoyl-1H-pyrrol-1-yl)propanoic acid 148

(E)-3-(carbamimidoylsulfanyl)prop-2-enoic acid 149

(Z)-3-(carbamimidoylsulfanyl)prop-2-enoic acid 150

3-(carbamimidoylsulfanyl)propanoic acid 151

3-(carbamothioylamino)propanoic acid 152

3-(carbamoylamino)propanoic acid 153

3-[(1,3-benzothiazol-2-yl)amino]propanoic acid 154

3-[(1,3-thiazol-2-yl)amino]propanoic acid 155

3-[(1H-1,3-benzodiazol-2-yl)amino]propanoic acid 156

3-[N-(2-aminobenzene-1-carbamimido)]propanoic acid 157

3-[(2-carbamimidoylphenyl)amino]propanoic acid 158

3-[(4,5-dihydro-1,3-thiazol-2-yl)amino]propanoic acid 159

3-[(6-ethyl-4-oxo-1,4-dihydropyrimidin-2- yl)amino]propanoic acid 160

3-[(9H-purin-6-yl)amino]propanoic acid 161

3-[(N-methylcarbamimidoyl)sulfanyl]propanoic acid 162

3-[(N,N-dimethylcarbamimidoyl)sulfanyl]propanoic acid 163

3-[(pyridin-2-yl)amino]propanoic acid 164

3-[(pyrimidin-2-yl)amino]propanoic acid 165

3-[2-cyanocarbamimidamido]propanoic acid 166

3-[2-nitrocarbamimidamido]propanoic acid 167

3-{[(acetylimino)(amino)methyl]amino}propanoic acid 168

3-{[(methylsulfanyl)methanimidoyl]amino}propanoic acid 169

3-{[amino[(ethoxycarbonyl)imino]methyl]amino}propanoic acid 173

3-carbamimidamido-2-(hydroxyimino)propanoic acid 174

3-carbamimidamido-2-(methylamino)propanoic acid 175

3-carbamimidamido-2-hydroxypropanoic acid 176

3-carbamimidamido-2-methylpropanoic acid 177

3-carbamimidamido-2-sulfanylpropanoic acid 178

3-carbamimidamido-3-phenylpropanoic acid 179

3-carbamimidamido-N-hydroxypropanamide 180

3-carbamimidamidooctanoic acid 181

3-carbamimidamidopropanamide 182

3-ethanimidamidopropanoic acid 183

4-(carbamimidoylsulfanyl)butanoic acid 184

4-carbamimidamidobenzoic acid 185

4-carbamimidoylbutanoic acid 186

ethyl 3-guanidinopropanoate 187

2-(2-imino-4-methylimidazolidin-1-yl)acetic acid 188

2-(2-imino-5-methylimidazolidin-1-yl)acetic acid 189

2-(2-imino-5-oxoimidazolidin-1-yl)acetic acid 190

2-(2-iminopyrrolidin-3-yl)acetic acid 191

2-{2-[(prop-2-en-1-yl)amino]-4,5-dihydro-1H-imidazol-1- yl}acetic acid192

2-[2-(methylamino)-4,5-dihydro-1H-imidazol-1-yl]acetic acid 193

2-[2-iminopyrrolidin-3-ylidene]acetic acid 194

2-imino-1,3-diazabicyclo[3.2.0]heptane-7-carboxylic acid 195

3-carbamimidoyl-3-methylprop-2-enoic acid 196

N-(benzenesulfonyl)-2-(1-methylguanidino)acetamide 197

[carbamimidoyl(methyl)carbamoyl]formic acid 198

2-(2-amino-5-methyl-6-oxo-1,6-dihydropyrimidin-1- yl)acetic acid 199

2-(3-imino-1,2,4-oxadiazolidin-4-yl)acetic acid 200

2-[({[(3- chlorophenyl)carbamoyl]amino}methanimidoyl)(methyl)amino]acetic acid 201

2-[1-(bromomethyl)guanidino]acetic acid 202

2-[1-methyl-2-(phosphonooxy)guanidino]acetic acid 203

2-[3-(carboxymethyl)-2-iminoimidazolidin-1-yl]acetic acid 204

2-{[amino(sulfoimino)methyl](methyl)amino}acetic acid 205

3-(1-methylguanidino)prop-2-enoic acid 206

3-(carbamimidoylsulfanyl)-2-methylpropanoic acid 207

3-(carbamimidoylsulfanyl)butanoic acid 209

4-carbamimidamidobut-2-enoic acid 210

1,3-diamino-2-iminoimidazolidin-4-one 211

3-amino-2-hydrazinylideneimidazolidin-4-one 212

3-hydrazinylidene-1,2,4-triazinan-6-one 213

2-(3-aminoguanidino)acetic acid 214

2-(3-nitroguanidino)acetic acid 215

2-(carbamimidamidoamino)propanoic acid 216

2-[3-(methylideneamino)guanidino]acetic acid 217

2-[1,2-diaminoguanidino]acetic acid 218

benzyl 2-(carbamimidamidoamino)acetate 323

2-oxoazetidine-1-carboximidamide 325

(2R,3R,4R,5R)-4-[(3-carbamimidamidopropanoyl)oxy]-5-(5-fluoro-2-oxo-4-{[(pentyloxy)carbonyl]amino}-1,2-dihydropyrimidin-1-yl)-2-methyloxolan-3-yl 3- carbamimidamidopropanoate326

1-[(3-carbamimidamidopropanoyl)oxy]-3-[({1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxo-1,2-dihydropyrimidin-4- yl}carbamoyl)oxy]propan-2-yl 3-carbamimidamidopropanoate 324

1-(pyridin-2-yl)azetidin-2-one 447

1-(pyridin-2-yl)azetidine-3-carboxylic acid 448

2-[1-(pyridin-2-yl)azetidin-3-yl]acetic acid

In another aspect, the invention features a compound selected from anyone of compounds 287-298 in Table 9.

TABLE 9 Selected Phosphocreatine System Inhibitors Compound NumberStructure Compound Name 287

(2S)-2-amino-3-{[{amino[(2- carboxyethyl)amino]methylidene}amino]sulfanyl}propanoic acid 288

(2S)-3-{[{amino[(2- carboxyethyl)amino]methylidene} amino]sulfanyl}-2-acetamidopropanoic acid 289

3-[2- [(carboxymethyl)sulfanyl] carbamimidamido]propanoic acid 290

3-[2-[(2- aminoethyl)sulfanyl] carbamimidamido]propanoic acid 291

(2R)-2-amino-4-{[{amino[(2- carboxyethyl)amino]methylidene}amino]sulfanyl}butanoic acid 292

3-[2-[(2- sulfoethyl)sulfanyl] carbamimidamido]propanoic acid 293

3-[2-{[(2S)-2-amino-2- carboxyethyl]sulfanyl} carbamimidamido]butanoicacid 294

3-[2-{[(2S)-2-carboxy-2- acetamidoethyl]sulfanyl}carbamimidamido]butanoic acid 295

3-[2- [(carboxymethyl)sulfanyl] carbamimidamidojbutanoic acid 296

3-[2-[(2- aminoethyl)sulfanyl] carbamimidamido]butanoic acid 297

(2R)-2-amino-4-{[{amino[(1- carboxypropan-2- yl)amino]methylidene}amino]sulfanyl}butanoic acid 298

3-[2-[(2- sulfoethyl)sulfanyl] carbamimidamido]butanoic acid

In another aspect, the invention features a compound selected from anyone of compounds 299-322 in Table 10.

TABLE 10 Selected Phosphocreatine System Inhibitors Compound NumberStructure Compound Name 299

(2S)-2-amino-3-({[1- (carboxymethyl)imidazolidin-2-ylidene]amino}sulfanyl)propanoic acid 300

(2S)-3-({[1- (carboxymethyl)imidazolidin-2- ylidene]amino}sulfanyl)-2-acetamidopropanoic acid 301

2-({[1- (carboxymethyl)imidazolidin-2- ylidene]amino}sulfanyl)aceticacid 302

2-[2-{[(2- aminoethyl)sulfanyl]imino} imidazolidin-1-yl]acetic acid 303

(2R)-2-amino-4-({[1- (carboxymethyl)imidazolidin-2-ylidene]amino}sulfanyl)butanoic acid 304

2-[2-{[(2- sulfoethyl)sulfanyl]imino} imidazolidin-1-yl]acetic acid 305

(2S)-2-amino-3-({[1- (carboxymethyl)-1,3-diazinan-2-ylidene]amino}sulfanyl)propanoic acid 306

(2S)-3-({[1-(carboxymethyl)-1,3- diazinan-2- ylidene]amino}sulfanyl)-2-acetamidopropanoic acid 307

2-({[1-(carboxymethyl)-1,3- diazinan-2- ylidene]amino}sulfanyl)aceticacid 308

2-[2-{[(2- aminoethyl)sulfanyl]imino}-1,3- diazinan-1-yl]acetic acid 309

(2R)-2-amino-4-({[1- (carboxymethyl)-1,3-diazinan-2-ylidene]amino}sulfanyl)butanoic acid 310

2-[2-{[(2- sulfoethyl)sulfanyl]imino}-1,3- diazinan-1-yl]acetic acid 311

(2S)-2-amino-3- {[{amino[(carboxymethyl)(methyl)amino]methylidene}amino] sulfanyl}propanoic acid 312

(2S)-3- {[{amino[(carboxymethyl)(methyl) amino]methylidene}amino]sulfanyl}-2-acetamidopropanoic acid 313

2- {[{[(carboxymethyl)(methyl)amino] (amino)methylidene}amino]sulfanyl}acetic acid 314

2-[2-[(2-aminoethyl)sulfanyl]-1- methylguanidino]acetic acid 315

(2R)-2-amino-4- {[{amino[(carboxymethyl)(methyl)amino]methylidene}amino] sulfanyl}butanoic acid 316

2-[1-methyl-2-[(2- sulfoethyl)sulfanyl]guanidino] acetic acid 317

(2S)-2-amino-3- {[{[(carboxymethyl)(methyl)amino](methylamino)methylidene} amino]sulfanyl}propanoic acid 318

(2S)-3- {[{[(carboxymethyl)(methyl)amino](methylamino)methylidene}amino] sulfanyl}-2-acetamidopropanoic acid 319

2- {[{[(carboxymethyl)(methyl)amino] (methylamino)methylidene}amino]sulfanyl}acetic acid 320

2-[2-[(2-aminoethyl)sulfanyl]-1,3- dimethylguanidino]acetic acid 321

(2R)-2-amino-4- {[{[(carboxymethyl)(methyl)amino](methylamino)methylidene} amino]sulfanyl}butanoic acid 322

2-[1,3-dimethyl-2-[(2- sulfoethyl)sulfanyl]guanidino] acetic acid

In another aspect, the invention features a composition comprising anyof the foregoing compounds and a pharmaceutically acceptable excipient.In some embodiments, the composition is a pharmaceutical composition. Insome embodiments, the creatine transport inhibitor or creatine kinaseinhibitor in the composition is substantially enantiomerically pure.

In another aspect, the invention features a method for treating cancer(e.g., gastrointestinal cancer such as, esophageal cancer, stomachcancer, pancreatic cancer, liver cancer, gallbladder cancer, colorectalcancer, anal cancer, mucosa-associated lymphoid tissue cancer,gastrointestinal stromal tumors, cancers of the biliary tree, andgastrointestinal carcioid tumor), comprising administering to a subjectin need thereof, any of the foregoing compounds in an amount sufficientto treat said cancer. In some embodiments, the compound is any of theforegoing compounds of Formula I, Formula II, or Formula III. In otherembodiments, the compound is any compound of any one of Tables 1-11(e.g., a compound of any one of Tables 1-7, 9 or 10).

TABLE 11 Selected Phosphocreatine System Inhibitors Compound NumberStructure Compound Name 106

5-amino-4- (aminomethyl)pentanoic acid 146

3-(1H-pyrazol-1-yl)propanoic acid 170

3-{4-amino-1H-pyrazolo[3,4- d]pyrimidin-1-yl}propanoic acid 171

3-{4-amino-2H-pyrazolo[3,4- d]pyrimidin-2-yl}propanoic acid 172

3-amino-1- (carboxylatomethyl)pyridin-1-ium 208

3-(carboxylatomethyl)-1- (carboxymethyl)-2-methyl-4,5-dihydro-1H-imidazol-3-ium

In another aspect, the invention features a method of slowing the spreadof a migrating cancer, including administering to a subject in needthereof, an inhibitor of creatine transport and/or creatine kinase in anamount sufficient to slow the spread of said migrating cancer. In someembodiments, the method comprises the suppression of metastaticcolonization of said migrating cancer in the liver of said subject. Insome embodiments, the migrating cancer is metastatic cancer (e.g.,including cells exhibiting migration, invasion of migrating cells,endothelial recruitment, and/or angiogenesis). In other embodiments, themigrating cancer spreads via seeding the surface of the peritoneal,pleural, pericardial, or subarachnoid spaces. In certain embodiments,the migrating cancer spreads via the lymphatic system. In someembodiments, the migrating cancer spreads hematogenously. In otherembodiments, the migrating cancer is a cell migration cancer (e.g., anon-metastatic cell migration cancer such as, ovarian cancer,mesothelioma, or primary lung cancer).

In another aspect, the invention features a method for inhibitingproliferation or growth of cancer stem cells or cancer initiating cells,including contacting the cell with an inhibitor of creatine transportand/or creatine kinase in an amount sufficient to inhibit proliferationor growth of said cell.

In another aspect, the invention features a method of reducing the rateof tumor seeding of a cancer including administering to a subject inneed thereof an inhibitor of creatine transport and/or creatine kinasein an amount sufficient to reduce tumor seeding.

In another aspect, the invention features a method of reducing ortreating metastatic nodule-forming of cancer including administering toa subject in need thereof an inhibitor of creatine transport and/orcreatine kinase in an amount sufficient to treat said metastaticnodule-forming of cancer.

In another aspect, the invention features a method of treatingmetastatic cancer in a subject in need thereof. The method includes: (a)providing a subject identified to have, or to be at risk of having,metastatic cancer on the basis of the expression level of miR-483-5pand/or miR-551a is below a predetermined reference value or theexpression level of CKB and/or SLC6a8 is above a predetermined referencevalue; and (b) administering to said subject an effective amount of anyof the foregoing compounds.

In another aspect, the invention features a method for treatingmetastatic cancer in a subject in need thereof, comprising contactingcreatine transport channel SLC6a8 with any of the foregoing compounds inan amount effective to suppress metastatic colonization of said cancer.

In some embodiments of any of the foregoing methods, the cancer isbreast cancer, colon cancer, renal cell cancer, non-small cell lungcancer, hepatocellular carcinoma, gastric cancer, ovarian cancer,pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, ormelanoma. In other embodiments of any of the foregoing methods thecancer is gastrointestinal cancer such as, esophageal cancer, stomachcancer, pancreatic cancer, liver cancer, gallbladder cancer, colorectalcancer, anal cancer, mucosa-associated lymphoid tissue cancer,gastrointestinal stromal tumors, cancers of the biliary tree, andgastrointestinal carcioid tumor.

In certain embodiments of any of the foregoing methods, the methodincludes administration of any of the foregoing compositions. In someembodiments of any of the foregoing methods, the method includesadministration of a composition including a creatine transport inhibitoror creatine kinase inhibitor that is substantially enantiomericallypure.

In other embodiments of any of the foregoing methods, the cancer is adrug resistant cancer (e.g., the cancer is resistant to vemurafenib,dacarbazine, a CTLA4 inhibitor, a BRAF inhibitor, a MEK inhibitor, a PD1inhibitor, or a PDL1 inhibitor).

In other embodiments of any of the foregoing methods, the method furtherincludes administering an antiproliferative (e.g., capecitabine,gemcitabine, fluorouracil, FOLFOX (5-FU, leucovorin, and Eloxatin),FOLFIRI (5-FU, leucovorin, and Camptosar), EOX (Epirubicin,Oxaliplatinum, and Xeloda), Taxotere, Erbitux, Zaltrap, Vectibix,Ramucirumab, Tivozanib, Stivarga, CRS-207, a PD-1 or PDL-1 antibody(e.g., nivolumab, pembrolizumab, MED14736, or MPDL3280A), and therapiesthat target CDK4/6, EGFR, PARP), wherein any of the foregoing compoundsand the antiproliferative are administered in an amount that together,is sufficient to slow the progression of migrating cancer. In certainembodiments, any of the foregoing compounds and the antiproliferativeare administered within 28 days of each other in amounts that togetherare effective to treat the subject.

In certain embodiments of any of the foregoing methods, the inhibitor ofcreatine transport and/or creatine kinase is any of the foregoingcompounds of Formula I, Formula II, Formula III, or Formula V. In otherembodiments, the compound is any compound of any one of Tables 1-11 or apharmaceutically acceptable salt thereof (e.g., a compound of any one ofTables 1-7, 9 or 10). In some embodiments, the compound is any compoundof Table 4 or a pharmaceutically acceptable salt thereof.

In some embodiments, of any of the foregoing methods, the inhibitor ofcreatine transport and/or creatine kinase is any one of compounds 7-9,11, 15, 16, 28, 29, 30, 32, 33, 34, 43, 47, 48, 51, 52, 54, 67, 69, 70,79, 80, 85, 124, 132, 149, 150, 187, 188, 190, 192-195, 199, 210-213,215, 217, 324, 447, or 448 or a pharmaceutically acceptable saltthereof. In other embodiments, of any of the foregoing methods, theinhibitor of creatine transport and/or creatine kinase is any one ofcompounds 16, 28, 29, 33, 34, 43, 47, 85, 124, 149, 150, 210-213, 215,217, 324, 447, or 448 or a pharmaceutically acceptable salt thereof. Incertain embodiments of any of the foregoing methods, the inhibitor ofcreatine transport and/or creatine kinase is any one of compounds 26,28, 34, 92, 96, 124, 125, 149, 150, 161, 163, 207, 324, 447 or 448 or apharmaceutically acceptable salt thereof. In certain embodiments of anyof the foregoing methods, the inhibitor of creatine transport and/orcreatine kinase is any one of compounds 28, 34, 124, 149, or 150 or apharmaceutically acceptable salt thereof. In some embodiments of any ofthe foregoing methods, the inhibitor of creatine transport and/orcreatine kinase is compound 34, a stereoisomer thereof, and/or apharmaceutically acceptable salt thereof. In some embodiments of any ofthe foregoing methods, the inhibitor of creatine transport and/orcreatine kinase is any one of compounds 219-224, or a pharmaceuticallyacceptable salt thereof.

Chemical Terms

As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins and C═Ndouble bonds can also be present in the compounds described herein, andall such stable isomers are contemplated in the present disclosure. Cisand trans geometric isomers of the compounds of the present disclosureare described and may be isolated as a mixture of isomers or asseparated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl. Herein a phrase of the form “optionally substituted X”(e.g., optionally substituted alkyl) is intended to be equivalent to “X,wherein X is optionally substituted” (e.g., “alkyl, wherein the alkyl isoptionally substituted”). It is not intended to mean that the feature“X” (e.g., alkyl) per se is optional.

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,trifluoroacetyl, propionyl, and butanoyl. Exemplary unsubstituted acylgroups include from 1 to 7, from 1 to 11, or from 1 to 21 carbons. Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein.

Non-limiting examples of optionally substituted acyl groups include,alkoxycarbonyl, alkoxycarbonylacyl, arylalkoxycarbonyl, aryloyl,carbamoyl, carboxyaldehyde, (heterocyclyl) imino, and (heterocyclyl)oyl:

The “alkoxycarbonyl” group, which as used herein, represents an alkoxy,as defined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The “alkoxycarbonylacyl” group, which as used herein, represents an acylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —C(O)-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylacyl include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ acyl, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ acyl, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ acyl). In someembodiments, each alkoxy and alkyl group is further independentlysubstituted with 1, 2, 3, or 4 substituents, as described herein (e.g.,a hydroxy group) for each group.

The “arylalkoxycarbonyl” group, which as used herein, represents anarylalkoxy group, as defined herein, attached to the parent moleculargroup through a carbonyl (e.g., —C(O)—O-alkyl-aryl). Exemplaryunsubstituted arylalkoxy groups include from 8 to 31 carbons (e.g., from8 to 17 or from 8 to 21 carbons, such as C₆₋₁₀ aryl-C₁₋₆alkoxy-carbonyl, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy-carbonyl, or C₆₋₁₀ aryl-C₁₋₂₀alkoxy-carbonyl). In some embodiments, the arylalkoxycarbonyl group canbe substituted with 1, 2, 3, or 4 substituents as defined herein.

The “aryloyl” group, which as used herein, represents an aryl group, asdefined herein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The “carbamoyl” group, which as used herein, represents—C(O)—N(R^(N1))₂, where the meaning of each R^(N1) is found in thedefinition of “amino” provided herein.

The “carboxyaldehyde” group, which as used herein, represents an acylgroup having the structure —CHO.

The “(heterocyclyl) imino” group, which as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The “(heterocyclyl)oyl” group, which as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through a carbonyl group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, and neopentyl, and may be optionally substituted with one,two, three, or, in the case of alkyl groups of two carbons or more, foursubstituents independently selected from the group consisting of: (1)C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclypoxy; (8) hydroxy,optionally substituted with an O-protecting group; (9) nitro; (10) oxo(e.g., carboxyaldehyde or acyl); (11) C₁₋₇, spirocyclyl; (12)thioalkoxy; (13) thiol; (14) —CO₂R^(A′), optionally substituted with anO-protecting group and where R^(A′) is selected from the groupconsisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(1′), wherein R^(N1) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R¹ is selected from the groupconsisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, and isopropylene. The term “C_(x-y)alkylene” and the prefix “C_(x-y) alk-” represent alkylene groups havingbetween x and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14,16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, or C₂₋₂₀alkylene). In some embodiments, the alkylene can be further substitutedwith 1, 2, 3, or 4 substituent groups as defined herein for an alkylgroup.

Non-limiting examples of optionally substituted alkyl and alkylenegroups include acylaminoalkyl, acyloxyalkyl, alkoxyalkyl,alkoxycarbonylalkyl, alkylsulfinyl, alkylsulfinylalkyl, aminoalkyl,carbamoylalkyl, carboxyalkyl, carboxyaminoalkyl, haloalkyl,hydroxyalkyl, perfluoroalkyl, and sulfoalkyl:

The “acylaminoalkyl” group, which as used herein, represents an acylgroup, as defined herein, attached to an amino group that is in turnattached to the parent molecular group through an alkylene group, asdefined herein (i.e., -alkyl-N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group (e.g.,haloalkyl) and R^(N1) is as defined herein). Exemplary unsubstitutedacylaminoalkyl groups include from 1 to 41 carbons (e.g., from 1 to 7,from 1 to 13, from 1 to 21, from 2 to 7, from 2 to 13, from 2 to 21, orfrom 2 to 41 carbons). In some embodiments, the alkylene group isfurther substituted with 1, 2, 3, or 4 substituents as described herein,and/or the amino group is —NH₂ or —NHR^(N1), wherein R^(N1) is,independently, OH, NO₂, NH₂, NR^(N2) ₂, SO₂R^(N2), SOR^(N2), alkyl,aryl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein),or alkoxycarbonylalkyl, and each R^(N2) can be H, alkyl, or aryl.

The “acyloxyalkyl” group, which as used herein, represents an acylgroup, as defined herein, attached to an oxygen atom that in turn isattached to the parent molecular group though an alkylene group (i.e.,-alkyl-O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxyalkyl groupsinclude from 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11carbons). In some embodiments, the alkylene group is, independently,further substituted with 1, 2, 3, or 4 substituents as described herein.

The “alkoxyalkyl” group, which as used herein, represents an alkyl groupthat is substituted with an alkoxy group. Exemplary unsubstitutedalkoxyalkyl groups include between 2 to 40 carbons (e.g., from 2 to 12or from 2 to 20 carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀ alkoxy-C₁₋₂₀ alkyl). In some embodiments,the alkyl and the alkoxy each can be further substituted with 1, 2, 3,or 4 substituent groups as defined herein for the respective group.

The “alkoxycarbonylalkyl” group, which as used herein, represents analkyl group, as defined herein, that is substituted with analkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)—OR, where Ris an optionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group).Exemplary unsubstituted alkoxycarbonylalkyl include from 3 to 41 carbons(e.g., from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3to 31 carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkyl, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). Insome embodiments, each alkyl and alkoxy group is further independentlysubstituted with 1, 2, 3, or 4 substituents as described herein (e.g., ahydroxy group).

The “alkylsulfinylalkyl” group, which as used herein, represents analkyl group, as defined herein, substituted with an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The “aminoalkyl” group, which as used herein, represents an alkyl group,as defined herein, substituted with an amino group, as defined herein.The alkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy, and/or an N-protecting group).

The “carbamoylalkyl” group, which as used herein, represents an alkylgroup, as defined herein, substituted with a carbamoyl group, as definedherein. The alkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The “carboxyalkyl” group, which as used herein, represents an alkylgroup, as defined herein, substituted with a carboxy group, as definedherein. The alkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein, and the carboxy group can beoptionally substituted with one or more O-protecting groups.

The “carboxyaminoalkyl” group, which as used herein, represents anaminoalkyl group, as defined herein, substituted with a carboxy, asdefined herein. The carboxy, alkyl, and amino each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as described hereinfor the respective group (e.g., CO₂R^(A′), where R^(A′) is selected fromthe group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen,and (d) C₁₋₆ alk-C₆₋₁₀ aryl, e.g., carboxy, and/or an N-protectinggroup, and/or an O-protecting group).

The “haloalkyl” group, which as used herein, represents an alkyl group,as defined herein, substituted with a halogen group (i.e., F, Cl, Br, orI). A haloalkyl may be substituted with one, two, three, or, in the caseof alkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCI₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The “hydroxyalkyl” group, which as used herein, represents an alkylgroup, as defined herein, substituted with one to three hydroxy groups,with the proviso that no more than one hydroxy group may be attached toa single carbon atom of the alkyl group, and is exemplified byhydroxymethyl and dihydroxypropyl. In some embodiments, the hydroxyalkylgroup can be substituted with 1, 2, 3, or 4 substituent groups (e.g.,O-protecting groups) as defined herein for an alkyl.

The “perfluoroalkyl” group, which as used herein, represents an alkylgroup, as defined herein, where each hydrogen radical bound to the alkylgroup has been replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl.

The “sulfoalkyl” group, which as used herein, represents an alkyl group,as defined herein, substituted with a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein, and the sulfo group can befurther substituted with one or more O-protecting groups (e.g., asdescribed herein).

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl.Alkenyls include both cis and trans isomers. Alkenyl groups may beoptionally substituted with 1, 2, 3, or 4 substituent groups that areselected, independently, from amino, aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

Non-limiting examples of optionally substituted alkenyl groups include,alkoxycarbonylalkenyl, aminoalkenyl, and hydroxyalkenyl:

The “alkoxycarbonylalkenyl” group, which as used herein, represents analkenyl group, as defined herein, that is substituted with analkoxycarbonyl group, as defined herein (e.g., -alkenyl-C(O)—OR, where Ris an optionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group).Exemplary unsubstituted alkoxycarbonylalkenyl include from 4 to 41carbons (e.g., from 4 to 10, from 4 to 13, from 4 to 17, from 4 to 21,or from 4 to 31 carbons, such as C₁₋₆ alkoxycarbonyl-C₂₋₆ alkenyl, C₁₋₁₀alkoxycarbonyl-C₂₋₁₀ alkenyl, or C₁₋₂₀ alkoxycarbonyl-C₂₋₂₀ alkenyl). Insome embodiments, each alkyl, alkenyl, and alkoxy group is furtherindependently substituted with 1, 2, 3, or 4 substituents as describedherein (e.g., a hydroxy group).

The “aminoalkenyl” group, which as used herein, represents an alkenylgroup, as defined herein, substituted with an amino group, as definedherein. The alkenyl and amino each can be further substituted with 1, 2,3, or 4 substituent groups as described herein for the respective group(e.g., CO₂R^(A′), where R^(A′) is selected from the group consisting of(a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀aryl, e.g., carboxy, and/or an N-protecting group).

The “hydroxyalkenyl” group, which as used herein, represents an alkenylgroup, as defined herein, substituted with one to three hydroxy groups,with the proviso that no more than one hydroxy group may be attached toa single carbon atom of the alkyl group, and is exemplified bydihydroxypropenyl, and hydroxyisopentenyl. In some embodiments, thehydroxyalkenyl group can be substituted with 1, 2, 3, or 4 substituentgroups (e.g., O-protecting groups) as defined herein for an alkyl.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, and 1-propynyl. Alkynyl groups may beoptionally substituted with 1, 2, 3, or 4 substituent groups that areselected, independently, from aryl, cycloalkyl, or heterocyclyl (e.g.,heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

Non-limiting examples of optionally substituted alkynyl groups includealkoxycarbonylalkynyl, aminoalkynyl, and hydroxyalkynyl:

The “alkoxycarbonylalkynyl” group, which as used herein, represents analkynyl group, as defined herein, that is substituted with analkoxycarbonyl group, as defined herein (e.g., -alkynyl-C(O)—OR, where Ris an optionally substituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group).Exemplary unsubstituted alkoxycarbonylalkynyl include from 4 to 41carbons (e.g., from 4 to 10, from 4 to 13, from 4 to 17, from 4 to 21,or from 4 to 31 carbons, such as C₁₋₆ alkoxycarbonyl-C₂₋₆ alkynyl, C₁₋₁₀alkoxycarbonyl-C₂₋₁₀ alkynyl, or C₁₋₂₀ alkoxycarbonyl-C₂₋₂₀ alkynyl). Insome embodiments, each alkyl, alkynyl, and alkoxy group is furtherindependently substituted with 1, 2, 3, or 4 substituents as describedherein (e.g., a hydroxy group).

The “aminoalkynyl” group, which as used herein, represents an alkynylgroup, as defined herein, substituted with an amino group, as definedherein. The alkynyl and amino each can be further substituted with 1, 2,3, or 4 substituent groups as described herein for the respective group(e.g., CO₂R^(A′), where R^(A′) is selected from the group consisting of(a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀aryl, e.g., carboxy, and/or an N-protecting group).

The “hydroxyalkynyl” group, which as used herein, represents an alkynylgroup, as defined herein, substituted with one to three hydroxy groups,with the proviso that no more than one hydroxy group may be attached toa single carbon atom of the alkyl group. In some embodiments, thehydroxyalkynyl group can be substituted with 1, 2, 3, or 4 substituentgroups (e.g., O-protecting groups) as defined herein for an alkyl.

The term “amidino,” as used herein, represents a group with thestructure

wherein each R^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), an N-protecting group, alkyl, alkenyl,alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl(e.g., optionally substituted with an O-protecting group, such asoptionally substituted arylalkoxycarbonyl groups or any describedherein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or othersdescribed herein), alkoxycarbonylalkyl (e.g., optionally substitutedwith an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), wherein each ofthese recited R^(N1) groups can be optionally substituted, as definedherein for each group; or two R^(N1) combine to form a heterocyclyl oran N-protecting group, and wherein each R^(N2) is, independently, H,alkyl, or aryl. Non-limiting examples of optionally substituted amidinogroups include guanidino, 2-amino-imidazoyl, 2-iminoimidazolidino,2-imino-1,3-diazinan-1-yl, 3-amino-1,2,4-triazol-4-yl, imidazol-2yl,1,4,5,6-tetrahydropyrimidin-2-yl, 1,2,4-triazol-3-yl,5-amino-3,4-dihydro-pyrrol-5-yl, imidazol-1-yl, carbamimidoylsulfanyl,carbamoylamino, carbamothioylamino, 2-amino-1,3-benzothiazol-2-yl,2-amino-1,3-thiazol-2-yl, 2-amino-1,3-benzodiazol-2-yl, and2-aminopyridyl.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionallysubstituted with an O-protecting group, such as optionally substitutedarylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl(e.g., acetyl, trifluoroacetyl, or others described herein),alkoxycarbonylalkyl (e.g., optionally substituted with an O-protectinggroup, such as optionally substituted arylalkoxycarbonyl groups or anydescribed herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl(e.g., alkheteroaryl), wherein each of these recited R^(N1) groups canbe optionally substituted, as defined herein for each group; or twoR^(N1) combine to form a heterocyclyl or an N-protecting group, andwherein each R^(N2) is, independently, H, alkyl, or aryl. The aminogroups of the invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others describedherein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, andeach R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

Non-limiting examples of optionally substituted amino groups includeacylamino and carbamyl:

The “acylamino” group, which as used herein, represents an acyl group,as defined herein, attached to the parent molecular group though anamino group, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H oran optionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group (e.g.,haloalkyl) and R^(N1) is as defined herein). Exemplary unsubstitutedacylamino groups include from 1 to 41 carbons (e.g., from 1 to 7, from 1to 13, from 1 to 21, from 2 to 7, from 2 to 13, from 2 to 21, or from 2to 41 carbons). In some embodiments, the alkyl group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein, and/orthe amino group is —NH₂ or —NHR^(N1), wherein R^(N1) is, independently,OH, NO₂, NH₂, NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, aryl,acyl (e.g., acetyl, trifluoroacetyl, or others described herein), oralkoxycarbonylalkyl, and each R^(N2) can be H, alkyl, or aryl.

The “carbamyl” group, which as used herein, refers to a carbamate grouphaving the structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where themeaning of each R^(N1) is found in the definition of “amino” providedherein, and R is alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), as defined herein.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇,spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R¹ is selected from the groupconsisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl(e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂), (CH₂)_(s3)OR′, wherein s1 is an integer from 1 to10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently,is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4,from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h2)amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, and indenyl, and may be optionally substituted with 1, 2, 3, 4,or 5 substituents independently selected from the group consisting of:(1) C₁₋₇, acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl(e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) Q₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The “arylalkyl” group, which as used herein, represents an aryl group,as defined herein, attached to the parent molecular group through analkylene group, as defined herein. Exemplary unsubstituted arylalkylgroups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20carbons, such as C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀alk-C₆₋₁₀ aryl). In some embodiments, the alkylene and the aryl each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective groups. Other groups preceded by the prefix“alk-” are defined in the same manner, where “alk” refers to a C₁₋₆alkylene, unless otherwise noted, and the attached chemical structure isas defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The term “boranyl,” as used herein, represents —B(R^(B1))₃, where eachR^(B1) is, independently, selected from the group consisting of H andoptionally substituted alkyl. In some embodiments, the boranyl group canbe substituted with 1, 2, 3, or 4 substituents as defined herein foralkyl.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxy,” as used herein, means —CO₂H.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and bicycle heptyl.When the cycloalkyl group includes one carbon-carbon double bond, thecycloalkyl group can be referred to as a “cycloalkenyl” group. Exemplarycycloalkenyl groups include cyclopentenyl, and cyclohexenyl. Thecycloalkyl groups of this invention can be optionally substituted with:(1) C₁₋₇, acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl(e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The “cycloalkylalkyl” group, which as used herein, represents acycloalkyl group, as defined herein, attached to the parent moleculargroup through an alkylene group, as defined herein (e.g., an alkylenegroup of from 1 to 4, from 1 to 6, from 1 to 10, or form 1 to 20carbons). In some embodiments, the alkylene and the cycloalkyl each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “heteroalkyl,” as used herein, refers to an alkyl group, asdefined herein, in which one or two of the constituent carbon atoms haveeach been replaced by nitrogen, oxygen, or sulfur. In some embodiments,the heteroalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups. The terms“heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl andalkynyl groups, as defined herein, respectively, in which one or two ofthe constituent carbon atoms have each been replaced by nitrogen,oxygen, or sulfur. In some embodiments, the heteroalkenyl andheteroalkynyl groups can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

Non-limiting examples of optionally substituted heteroalkyl,heteroalkenyl, and heteroalkynyl groups include acyloxy, alkenyloxy,alkoxy, alkoxyalkoxy, alkoxycarbonylalkoxy, alkynyloxy, aminoalkoxy,arylalkoxy, carboxyalkoxy, cycloalkoxy, haloalkoxy, (heterocyclyl)oxy,perfluoroalkoxy, thioalkoxy, and thioheterocyclylalkyl:

The “acyloxy” group, which as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an oxygenatom (i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆,C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groupsinclude from 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11carbons). In some embodiments, the alkyl group is further substitutedwith 1, 2, 3, or 4 substituents as described herein.

The “alkenyloxy” group, which as used here, represents a chemicalsubstituent of formula —OR, where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆or C₂₋₁₀ alkenyl), unless otherwise specified. Exemplary alkenyloxygroups include ethenyloxy, and propenyloxy. In some embodiments, thealkenyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as defined herein (e.g., a hydroxy group).

The “alkoxy” group, which as used herein, represents a chemicalsubstituent of formula —OR, where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆or C₁₋₁₀ alkyl), unless otherwise specified. Exemplary alkoxy groupsinclude methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), andt-butoxy. In some embodiments, the alkyl group can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein(e.g., hydroxy or alkoxy).

The “alkoxyalkoxy” group, which as used herein, represents an alkoxygroup that is substituted with an alkoxy group. Exemplary unsubstitutedalkoxyalkoxy groups include between 2 to 40 carbons (e.g., from 2 to 12or from 2 to 20 carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀alkoxy-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments,the each alkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as defined herein.

The “alkoxycarbonylalkoxy” group, which as used herein, represents analkoxy group, as defined herein, that is substituted with analkoxycarbonyl group, as defined herein (e.g., —O-alkyl-C(O)—OR, where Ris an optionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group).Exemplary unsubstituted alkoxycarbonylalkoxy include from 3 to 41carbons (e.g., from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21,or from 3 to 31 carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The “alkynyloxy” group, which as used herein, represents a chemicalsubstituent of formula —OR, where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆or C₂₋₁₀ alkynyl), unless otherwise specified. Exemplary alkynyloxygroups include ethynyloxy, and propynyloxy. In some embodiments, thealkynyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as defined herein (e.g., a hydroxy group).

The “aminoalkoxy” group, which as used herein, represents an alkoxygroup, as defined herein, substituted with an amino group, as definedherein. The alkyl and amino each can be further substituted with 1, 2,3, or 4 substituent groups as described herein for the respective group(e.g., CO₂R^(A′), where R^(A′) is selected from the group consisting of(a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀aryl, e.g., carboxy).

The “arylalkoxy” group, which as used herein, represents an alkarylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. Exemplary unsubstituted arylalkoxy groups include from 7to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein.

The “aryloxy” group, which as used herein, represents a chemicalsubstituent of formula —OR′, where R′ is an aryl group of 6 to 18carbons, unless otherwise specified. In some embodiments, the aryl groupcan be substituted with 1, 2, 3, or 4 substituents as defined herein.

The “carboxyalkoxy” group, which as used herein, represents an alkoxygroup, as defined herein, substituted with a carboxy group, as definedherein. The alkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the alkyl group, and thecarboxy group can be optionally substituted with one or moreO-protecting groups.

The “cycloalkoxy” group, which as used herein, represents a chemicalsubstituent of formula —OR, where R is a C₃₋₈ cycloalkyl group, asdefined herein, unless otherwise specified. The cycloalkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as describedherein. Exemplary unsubstituted cycloalkoxy groups are from 3 to 8carbons. In some embodiment, the cycloalkyl group can be furthersubstituted with 1, 2, 3, or 4 substituent groups as described herein.

The “haloalkoxy” group, which as used herein, represents an alkoxygroup, as defined herein, substituted with a halogen group (i.e., F, Cl,Br, or I). A haloalkoxy may be substituted with one, two, three, or, inthe case of alkyl groups of two carbons or more, four halogens.Haloalkoxy groups include perfluoroalkoxys (e.g., —OCF₃), —OCHF₂,—OCH₂F, —OCCl₃, —OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHlCH₃. In someembodiments, the haloalkoxy group can be further substituted with 1, 2,3, or 4 substituent groups as described herein for alkyl groups.

The “(heterocyclyl)oxy” group, which as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an oxygen atom. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The “perfluoroalkoxy” group, which as used herein, represents an alkoxygroup, as defined herein, where each hydrogen radical bound to thealkoxy group has been replaced by a fluoride radical. Perfluoroalkoxygroups are exemplified by trifluoromethoxy and pentafluoroethoxy.

The “alkylsulfinyl” group, which as used herein, represents an alkylgroup attached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The “thioarylalkyl” group, which as used herein, represents a chemicalsubstituent of formula —SR, where R is an arylalkyl group. In someembodiments, the arylalkyl group can be further substituted with 1, 2,3, or 4 substituent groups as described herein.

The “thioalkoxy” group as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The “thioheterocyclylalkyl” group, which as used herein, represents achemical substituent of formula —SR, where R is an heterocyclylalkylgroup. In some embodiments, the heterocyclylalkyl group can be furthersubstituted with 1, 2, 3, or 4 substituent groups as described herein.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heteroarylalkyl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted heteroarylalkyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, andbenzothienyl. Examples of fused heterocyclyls include tropanes and1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, and benzothienyl, including dihydro and tetrahydroforms thereof, where one or more double bonds are reduced and replacedwith hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl;2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl;3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(21-1)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇, acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The “heterocyclylalkyl” group, which as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an alkylene group, as defined herein. Exemplaryunsubstituted heterocyclylalkyl groups are from 2 to 32 carbons (e.g.,from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15,from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C₁₋₆alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂ heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂heterocyclyl). In some embodiments, the alkylene and the heterocyclyleach can be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group. In someembodiments, the hydroxy group can be substituted with 1, 2, 3, or 4substituent groups (e.g., O-protecting groups) as defined herein for analkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, and phenylalanine; sulfonyl-containing groups such asbenzenesulfonyl, and p-toluenesulfonyl; carbamate forming groups such asbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl,alkaryl groups such as benzyl, triphenylmethyl, and benzyloxymethyl, andsilyl groups, such as trimethylsilyl. Preferred N-protecting groups areformyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl,phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl(Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “O-protecting group,” as used herein, represents those groupsintended to protect an oxygen containing (e.g., phenol, hydroxyl, orcarbonyl) group against undesirable reactions during syntheticprocedures. Commonly used O-protecting groups are disclosed in Greene,“Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley &Sons, New York, 1999), which is incorporated herein by reference.Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups,such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl,tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl,phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and4-nitrobenzoyl; alkylcarbonyl groups, such as acyl, acetyl, propionyl,and pivaloyl; optionally substituted arylcarbonyl groups, such asbenzoyl; silyl groups, such as trimethylsilyl (TMS),tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), andtriisopropylsilyl (TIPS); ether-forming groups with the hydroxyl, suchmethyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, andtrityl; alkoxycarbonyls, such as methoxycarbonyl, ethoxycarbonyl,isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl,isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl,2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, and methyloxycarbonyl;alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl,ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl,2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl,allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, and3-methyl-2-butenoxycarbonyl; haloalkoxycarbonyls, such as2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, and2,2,2-trichloroethoxycarbonyl; optionally substituted arylalkoxycarbonylgroups, such as benzyloxycarbonyl, p-methylbenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-bromobenzyloxy-carbonyl, andfluorenylmethyloxycarbonyl; and optionally substituted aryloxycarbonylgroups, such as phenoxycarbonyl, p-nitrophenoxycarbonyl,o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl,p-methyl-phenoxycarbonyl, m-methylphenoxycarbonyl,o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl,p-chlorophenoxycarbonyl, and 2-chloro-4-nitrophenoxy-carbonyl);substituted alkyl, aryl, and alkaryl ethers (e.g., trityl;methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl;2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl;ethoxyethyl; 1-[2-(trimethylsilypethoxy]ethyl; 2-trimethylsilylethyl;t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl,p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl;triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl;t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl;triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl,methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl;2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl;methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl);carbonyl-protecting groups (e.g., acetal and ketal groups, such asdimethyl acetal, and 1,3-dioxolane; acylal groups; and dithiane groups,such as 1,3-dithianes, and 1,3-dithiolane); carboxylic acid-protectinggroups (e.g., ester groups, such as methyl ester, benzyl ester, t-butylester, and orthoesters; and oxazoline groups.

Exemplary O- and N-protecting groups include: Acetyl (Ac); Acylals;Benzoyl (Bz); Benzyl (Bn, BnI); Benzyl esters; Carbamate; Carbobenzyloxy(Cbz); Dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT);Dithianes; Ethoxyethyl ethers (EE); Methoxymethyl ether (MOM);Methoxytrityl [(4-methoxyphenyl)diphenylmethyl], (MMT); Methyl Ethers;Methyl (Me); Methyl esters; Methylthiomethyl ether; Orthoesters;Oxazoline; Pivaloyl (Piv); Phthalimido; p-Methoxybenzyl carbonyl (Moz orMeOZ); p-Methoxybenzyl (PMB); Propargyl alcohols; Silyl groups (e.g.,trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS)); Silylesters; tert-Butyl esters; tert-Butyloxycarbonyl (Boc or tBoc);Tetrahydropyranyl (THP); Tosyl (Ts or Tos); Trimethylsilylethoxymethyl(SEM); Trityl (triphenylmethyl, Tr); β-Methoxyethoxymethyl ether (MEM);(4-Nitrophenyl)sulfonyl or (4-nitrophenyl)(dioxido)-lambda(6)-sulfanyl)(Nosyl); 2-Cyanoethyl; 2-Nitrophenylsulfenyl (Nps); 3,4-Dimethoxybenzyl(DMPM); and 9-Fluorenylmethyloxycarbonyl (FMOC)

The term “oxo” as used herein, represents ═O.

The prefix “perfluoro,” as used herein, represents anyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. For example, perfluoroalkyl groupsare exemplified by trifluoromethyl, and pentafluoroethyl.

The term “protected hydroxyl,” as used herein, refers to an oxygen atombound to an O-protecting group.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group. Theterm “thiol,” as used herein represents an —SH group.

Definitions

The term “cancer” refers to any cancer caused by the proliferation ofmalignant neoplastic cells, such as tumors, neoplasms, carcinomas,sarcomas, leukimias, and lymphomas.

“Cell migration” as used in this application involves the invasion bythe cancer cells into the surrounding tissue and the crossing of thevessel wall to exit the vasculature in distal organs of the cancer cell.

By “cell migration cancers” is meant cancers that migrate by invasion bythe cancer cells into the surrounding tissue and the crossing of thevessel wall to exit the vasculature in distal organs of the cancer cell.

As used herein, “drug resistant cancer” refers to any cancer that isresistant to an antiproliferative in Table 11.

As used herein, “metastatic nodule” refers to an aggregation of tumorcells in the body at a site other than the site of the original tumor.

As used herein, “metastatic tumor” refers to a tumor or cancer in whichthe cancer cells forming the tumor have a high potential to or havebegun to, metastasize, or spread from one location to another locationor locations within a subject, via the lymphatic system or viahaematogenous spread, for example, creating secondary tumors within thesubject. Such metastatic behavior may be indicative of malignant tumors.In some cases, metastatic behavior may be associated with an increase incell migration and/or invasion behavior of the tumor cells.

Examples of cancers that can be defined as metastatic include but arenot limited to non-small cell lung cancer, breast cancer, ovariancancer, colorectal cancer, biliary tract cancer, bladder cancer, braincancer including glioblastomas and medullablastomas, cervical cancer,choriocarcinoma, endometrial cancer, esophageal cancer, gastric cancer,hematological neoplasms, multiple myeloma, leukemia, intraepithelialneoplasms, livercancer, lymphomas, neuroblastomas, oral cancer,pancreatic cancer, prostate cancer, sarcoma, skin cancer includingmelanoma, basocellular cancer, squamous cell cancer, testicular cancer,stromal tumors, germ cell tumors, thyroid cancer, and renal cancer.

As used herein, “migrating cancer” refers to a cancer in which thecancer cells forming the tumor migrate and subsequently grow asmalignant implants at a site other than the site of the original tumor.The cancer cells migrate via seeding the surface of the peritoneal,pleural, pericardial, or subarachnoid spaces to spread into the bodycavities; via invasion of the lymphatic system through invasion oflymphatic cells and transport to regional and distant lymph nodes andthen to other parts of the body; via haematogenous spread throughinvasion of blood cells; or via invasion of the surrounding tissue.Migrating cancers include metastatic tumors and cell migration cancers,such as ovarian cancer, mesothelioma, and primary lung cancer, each ofwhich is characterized by cellular migration.

“Non-metastatic cell migration cancer” as used herein refers to cancersthat do not migrate via the lymphatic system or via haematogenousspread.

“Proliferation” as used in this application involves reproduction ormultiplication of similar forms (cells) due to constituting (cellular)elements.

As used herein, “slowing the spread of metastasis” refers to reducing orstopping the formation of new loci; or reducing, stopping, or reversingthe tumor load.

As used herein, “slowing the spread of migrating cancer” refers toreducing or stopping the formation of new loci; or reducing, stopping,or reversing the tumor load.

As used herein “substantially enantiomerically pure,” refers to acomposition (e.g., a pharmaceutical composition) wherein greater 85%(e.g., greater than 90%, greater than 95%, up to and including 100%,i.e., within the limits of detection) of the molecules of creatinetransport inhibitor or creatine kinase inhibitor in the composition havethe same chirality sense.

As used herein, and as well understood in the art, “to treat” acondition or “treatment” of the condition (e.g., the conditionsdescribed herein such as cancer) is an approach for obtaining beneficialor desired results, such as clinical results. Beneficial or desiredresults can include, but are not limited to, alleviation or ameliorationof one or more symptoms or conditions; diminishment of extent ofdisease, disorder, or condition; stabilized (i.e., not worsening) stateof disease, disorder, or condition; preventing spread of disease,disorder, or condition; delay or slowing the progress of the disease,disorder, or condition; amelioration or palliation of the disease,disorder, or condition; and remission (whether partial or total),whether detectable or undetectable. “Palliating” a disease, disorder, orcondition means that the extent and/or undesirable clinicalmanifestations of the disease, disorder, or condition are lessenedand/or time course of the progression is slowed or lengthened, ascompared to the extent or time course in the absence of treatment.

As used herein, “tumor seeding” refers to the spillage of tumor cellclusters and their subsequent growth as malignant implants at a siteother than the site of the original tumor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are a set of diagrams and photographs showing thatmiR-483-5p, miR-551a and CKB are clinically relevant and can betherapeutically inhibited. a, miR-483-5p and miR-551a levels in 37primary tumor samples and 30 liver metastases samples were quantified byquantitative real-time PCR. b, CKB expression levels in 37 primary tumorsamples and 30 liver metastases samples were measured by quantitativereal-time PCR. c, Liver metastasis in mice injected with LvM3b cells andtreated with a single dose of AAV doubly expressing miR-483-5p andmiR-551a one day after injection cells. d, Bioluminescent measurementsof liver metastasis in mice injected with 5×10⁵ LvM3b cells and treatedwith cyclocreatine daily for two weeks. Mice were euthanized and liversexcised for ex vivo imaging at the end of the treatment. e,Bioluminescent measurements of liver metastasis in mice injected with5×10⁵ LvM3b cells and treated with the creatine transporter inhibitorbeta-guanidinopropionic acid ((β-GPA) daily for two weeks. Error bars,s.e.m; all P values are based on one-sided Student's t-tests. *P<0.05;**P<0.001; ***P<0.0001.

FIG. 2 is a diagram and a photograph showing that β-GPA treatmentsuppressed colorectal cancer metastasis. Bioluminescent measurements ofliver metastasis in mice injected with 5×10⁵ LvM3b cells and treatedwith β-GPA daily for three weeks. Mice were euthanized at three weeksand liver extracted for bioluminescent imaging and gross histology.Error bars represent the s.e.m; all P values are based on one-sidedStudent's t-tests. *P<0.05.

FIGS. 3A-C are a set of diagrams and photographs showing that creatinetransporter, SLC6a8 is required for colorectal and pancreatic cancermetastasis. a) Liver metastasis by highly aggressive LvM3b cellsexpressing short hairpins targeting the creatine transporter channel,SLC6a8. Liver metastasis were monitored by bioluminescent imaging andmice were euthanized three weeks after inoculation of cancer cells.Livers were extracted for gross histology. b) Liver metastasis in miceinjected with 5×10⁵ SW480 cells transduced with a shRNA targetingSLC6a8. Metastatic progression was monitored by bioluminescent imaging.Mice were euthanized 28 days after injection and livers excised forbioluminescent imaging and gross histology. c) Liver metastasis in miceinjected with 5×10⁵ PANC1 pancreatic cancer cells transduced with ashRNA targeting SLC6a8. Metastatic progression was monitored bybioluminescent imaging and mice were euthanized as described above.Error bars represent the s.e.m; all P values are based on one-sidedStudent's t-tests. *P<0.05; **P<0.001; ***P<0.0001.

FIG. 4 is a diagram showing that SLC6a8 is up-regulated in livermetastases compared to primary tumors. Expression of SLC6a8 in 36primary tumors and 30 liver metastases were quantified by quantitativereal-time PCR. Error bars represent the s.e.m; all P values are based onone-sided Student's t-tests. *P<0.05.

FIG. 5 is a diagram and a photograph showing that β-GPA treatmentsuppresses survival of disseminated PANC1 pancreatic cancer cells in theliver in vivo. Bioluminescence imaging of immunodeficient mice injectedwith 5×10⁵ PANC1 cells with and without 10 mM β-GPA-pre-treatment for 48hr. Mice were imaged on day 1 after injection and signal was normalizedto day zero. P values are based on one-sided Student's t-tests. *P<0.05.

FIG. 6 is a diagram showing that β-GPA enhances the cytotoxicity ofGemcitabine on PANC1 pancreatic cancer cells. Cell viability of PANC1pancreatic cancer cells after treatment with escalating doses ofGemcitabine alone or escalating doses of Gemcitabine in combination with10 mM β-GPA. Cell viability was assayed using the WST-1 reagent. Errorbars represent standard error of the mean.

FIG. 7 is a diagram showing that β-GPA enhances the cytotoxicity of5′-fluorouracil on LS-LvM3b colorectal cancer cells. Cell viability ofLs-LvM3b cells after treatment with escalating doses of 5′-Fluorouracilalone or escalating doses of 5′-Fluorouracil in combination with 10 mMβ-GPA. Cell viability was assayed using the WST-1 reagent. Error barsrepresent standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features methods for preventing or reducingaberrant proliferation, differentiation, or survival of cells. Forexample, compounds of the invention may be useful in reducing the riskof, or preventing, tumors from increasing in size or from reaching ametastatic state. The subject compounds may be administered to halt theprogression or advancement of cancer. In addition, the instant inventionincludes use of the subject compounds to reduce the risk of, or prevent,a recurrence of cancer.

Compounds

The invention features compounds useful in the treatment of cancer.Exemplary compounds described herein include compounds having astructure according to Formulae I-Ill as described herein:

wherein X¹ is absent, NH, or CH₂;

R¹ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R², R³, and R⁴ are independently hydrogen or optionally substitutedC₁-C₆ alkyl; and

R⁵ and R⁶ are hydrogen or NH₂;

wherein if R⁵ and R⁶ are both hydrogen or R⁵ is NH₂ and R⁶ is hydrogenthen R² is optionally substituted C₁-C₆ alkyl,

or a pharmaceutically acceptable salt thereof;

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl;

X² is S or NR¹²;

m is 0 or 1;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁵ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, NH₂,optionally substituted C₁-C₆ alkyl, or R⁵ or R⁹ can combine with R¹⁰ orR¹¹ to form an optionally substituted C₃-C₆ cycloalkyl ring or with R¹²to form an optionally substituted C₃-C₆ heterocycle;

R¹° and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₆ alkyl, or R¹⁰ or R¹¹ can combine with R⁵ or R⁹ to forman optionally substituted C₃-C₆ cycloalkyl ring;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² can combinewith R⁵ or R⁹ to form an optionally substituted C₃-C₆ heterocycle, and

wherein if R⁹ is halo then R⁵ is halo or optionally substituted C₁-C₆alkyl,

or a pharmaceutically acceptable salt thereof;

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl; m is 1 or 2;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁸ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, NH₂,optionally substituted C₁-C₃ alkyl, or R⁸ and R⁹ combine with the atomsto which they are attached to form an optionally substituted

C₃-C₆ cycloalkyl ring; or R⁸ or R⁹ combine with R¹³ or R¹¹ with theatoms to which they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R⁸ or R⁹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₅ heterocycle;

R¹³ and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₄ alkyl, optionally substituted C₂-C₆ alkenyl,optionally substituted C₂-C₆ alkynyl or R¹³ and R¹¹ combine with theatoms to which they are attached to form an optionally substituted C₃-C₆cycloalkyl ring; or R¹³ or R¹¹ combine with R⁸ or R⁹ with the atoms towhich they are attached to form an optionally substituted C₃-C₄cycloalkyl ring; or R¹³ or R¹¹ combine with R¹² with the atoms to whichthey are attached to form an optionally substituted C₃-C₄ heterocycle;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² combineswith R⁸ or R⁹ with the atoms to which they are attached to form anoptionally substituted C₃-C₅ heterocycle, or R¹² combines with R¹⁰ orR¹¹ with the atoms to which they are attached to form an optionallysubstituted C₃-C₄ heterocycle

wherein if m is 1 and R⁸ is hydrogen, halo, hydroxyl, or methyl then atleast one of R⁹, R¹⁰, and R¹¹ is not hydrogen;

wherein if m is 1 and R¹³ is methyl then at least one of R⁸, R⁹, and R¹¹is not hydrogen;

wherein if m is 1 and R⁸ is NH₂ and R¹³ is hydrogen, methyl, or—CH₂CH₂OH then at least one of R⁹ or R¹¹ is not hydrogen;

wherein if m is 1, R⁸ is halo, and R¹³ is optionally substituted C₁-C₄alkyl then at least one of R⁹ and R¹³ is not hydrogen;

or a pharmaceutically acceptable salt thereof; andA-B  Formula III

wherein A is a inhibitor of creatine transport and/or creatine kinasecomprising an amidino group; B has the structure:

wherein n is 0 or 1;

Q² is hydroxyl, optionally substituted amino, or —SO₂OH; and

R¹³ and R¹⁴ are independently hydrogen, —CO₂H, or combine to form C═O;

wherein B is conjugated to A at one of the amidino nitrogens,

or a pharmaceutically acceptable salt thereof.

Other embodiments (e.g., Compounds 1-326 of Tables 1-11) as well asexemplary methods for the synthesis of these compounds are describedherein.

Utility and Administration

The compounds described herein (e.g., a compound according to FormulaeI-IX or any of Compounds 1-448 of Tables 1-11) are useful in the methodsof the invention and, while not bound by theory, are believed to exerttheir desirable effects through their ability to inhibit creatinetransport and/or creatine kinase. The compounds described herein (e.g.,a compound according to Formulae I-IX or any of Compounds 1-448 ofTables 1-11) can also be used for the treatment of certain conditionssuch as cancer.

Creatine helps supply energy to all cells in the body by increasingformation of ATP. It is taken up by tissues with high energy demandsthrough an active transport system. The conversion of ADP to ATP byphosphate transfer from phosphocreatine is catylzed by creatine kinase.Some of the functions associated with the phosphocreatine system includeefficient regeneration of energy in the form of ATP in cells withfluctuating and high energy demand, energy transport to different partsof the cell, phosphoryl transport activity, ion transport regulation,and involvement in signal transduction pathways.

Creatine kinase has been shown to have elevated levels in certain tumortypes. These tumor types may utilize the increased expression ofcreatine kinase to prevent apoptosis under hypoxic or hypoglycemicconditions. Malignant cancers with poor prognosis have also been shownto overexpress creatine kinases, which may be related to high energyturnover and failure to eliminate cancer cells by apoptosis. Inhibtionof the active transport of creatine into cancer cells may reverse thesetrends and result in inhibition of the cancer and/or metastasis.

Treatment Methods

As disclosed herein, inhibition of creatine transport and/or creatinekinase suppresses metastasis. The phosphocreatine system promotesmetastasis by enhancing the survival of disseminated cancer cells in theliver by acting as an energetic store for ATP generation to endurehepatic hypoxia. Inhibition of creatine transport into cancer cellslimits the amount of phosphocreatine available to use in the productionof ATP. Inhibition of creatine kinase inhibits the production of ATPthrough conversion of phosphocreatine to creatine.

Typical vascularized tumors that can be treated with the method includesolid tumors, particularly carcinomas, which require a vascularcomponent for the provision of oxygen and nutrients. Exemplary solidtumors include, but are not limited to, carcinomas of the lung, breast,bone, ovary, stomach, pancreas, larynx, esophagus, testes, liver,parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,kidney, bladder, prostate, thyroid, squamous cell carcinomas,adenocarcinomas, small cell carcinomas, melanomas, gliomas,glioblastomas, neuroblastomas, Kaposi's sarcoma, and sarcomas.

Treating cancer can result in a reduction in size or volume of a tumor.For example, after treatment, tumor size is reduced by 5% or greater(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relativeto its size prior to treatment. Size of a tumor may be measured by anyreproducible means of measurement. The size of a tumor may be measuredas a diameter of the tumor or by any reproducible means of measurement.

Treating cancer may further result in a decrease in number of tumors.For example, after treatment, tumor number is reduced by 5% or greater(e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relativeto number prior to treatment. Number of tumors may be measured by anyreproducible means of measurement. The number of tumors may be measuredby counting tumors visible to the naked eye or at a specifiedmagnification (e.g., 2×, 3×, 4×, 5×, 10×, or 50×).

Treating cancer can result in a decrease in number of metastatic nodulesin other tissues or organs distant from the primary tumor site. Forexample, after treatment, the number of metastatic nodules is reduced by5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orgreater) relative to number prior to treatment. The number of metastaticnoduless may be measured by any reproducible means of measurement. Thenumber of metastatic nodules may be measured by counting metastaticnodules visible to the naked eye or at a specified magnification (e.g.,2×, 10×, or 50×).

Treating cancer can result in an increase in average survival time of apopulation of subjects treated according to the present invention incomparison to a population of untreated subjects. For example, theaverage survival time is increased by more than 30 days (more than 60days, 90 days, or 120 days). An increase in average survival time of apopulation may be measured by any reproducible means. An increase inaverage survival time of a population may be measured, for example, bycalculating for a population the average length of survival followinginitiation of treatment with the compound of the invention. An increasein average survival time of a population may also be measured, forexample, by calculating for a population the average length of survivalfollowing completion of a first round of treatment with the compound ofthe invention.

Treating cancer can also result in a decrease in the mortality rate of apopulation of treated subjects in comparison to an untreated population.For example, the mortality rate is decreased by more than 2% (e.g., morethan 5%, 10%, or 25%). A decrease in the mortality rate of a populationof treated subjects may be measured by any reproducible means, forexample, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith the compound of the invention. A decrease in the mortality rate ofa population may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with the compound ofthe invention.

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the therapeutic agents describedabove. The composition can be a pharmaceutical composition that containsa pharmaceutically acceptable carrier, a dietary composition thatcontains a dietarily acceptable suitable carrier, or a cosmeticcomposition that contains a cosmetically acceptable carrier. The term“pharmaceutical composition” refers to the combination of an activeagent with a carrier, inert or active, making the composition especiallysuitable for diagnostic or therapeutic use in vivo or ex vivo. A“pharmaceutically acceptable carrier,” after administered to or upon asubject, does not cause undesirable physiological effects. The carrierin the pharmaceutical composition must be “acceptable” also in the sensethat it is compatible with the active ingredient and can be capable ofstabilizing it. One or more solubilizing agents can be utilized aspharmaceutical carriers for delivery of an active compound. Examples ofa pharmaceutically acceptable carrier include, but are not limited to,biocompatible vehicles, adjuvants, additives, and diluents to achieve acomposition usable as a dosage form. Examples of other carriers includecolloidal silicon oxide, magnesium stearate, cellulose, sodium laurylsulfate, and D&C Yellow #10.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, or allergic response, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts of amines, carboxylic acids, and other types ofcompounds, are well known in the art. For example, S. M. Berge, et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting a free base or free acid function with a suitable reagent, asdescribed generally below. For example, a free base function can bereacted with a suitable acid. Furthermore, where the compounds of theinvention carry an acidic moiety, suitable pharmaceutically acceptablesalts thereof may, include metal salts such as alkali metal salts, e.g.sodium or potassium salts; and alkaline earth metal salts, e.g. calciumor magnesium salts. Examples of pharmaceutically acceptable, nontoxicacid addition salts are salts of an amino group formed with inorganicacids such as hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid and perchloric acid or with organic acids such as aceticacid, oxalic acid, maleic acid, tartaric acid, citric acid, succinicacid or malonic acid or by using other methods used in the art such asion exchange. Other pharmaceutically acceptable salts, include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, and valerate salts. Representative alkali or alkaline earthmetal salts include sodium, lithium, potassium, calcium, and magnesium.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutical compositions of the presentinvention additionally include a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, and lubricants, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; natural and synthetic phospholipids, such as soybean andegg yolk phosphatides, lecithin, hydrogenated soy lecithin, dimyristoyllecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleoyl lecithin,hydroxylated lecithin, lysophosphatidylcholine, cardiolipin,sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine,diastearoyl phosphatidylethanolamine (DSPE) and its pegylated esters,such as DSPE-PEG750 and, DSPE-PEG2000, phosphatidic acid, phosphatidylglycerol and phosphatidyl serine. Commercial grades of lecithin whichare preferred include those which are available under the trade namePhosal® or Phospholipon® and include Phosal 53 MCT, Phosal 50 PG, Phosal75 SA, Phospholipon 90H, Phospholipon 90G and Phospholipon 90 NG;soy-phosphatidylcholine (SoyPC) and DSPE-PEG2000 are particularlypreferred; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The above-described composition, in any of the forms described above,can be used for treating melanoma, or any other disease or conditiondescribed herein. An effective amount refers to the amount of an activecompound/agent that is required to confer a therapeutic effect on atreated subject. Effective doses will vary, as recognized by thoseskilled in the art, depending on the types of diseases treated, route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatment. A pharmaceutical composition of thisinvention can be administered parenterally, orally, nasally, rectally,topically, or buccally. The term “parenteral” as used herein refers tosubcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, or intracranial injection, as well as any suitableinfusion technique.

A sterile injectable composition can be a solution or suspension in anon-toxic parenterally acceptable diluent or solvent. Such solutionsinclude, but are not limited to, 1,3-butanediol, mannitol, water,Ringer's solution, and isotonic sodium chloride solution. In addition,fixed oils are conventionally employed as a solvent or suspending medium(e.g., synthetic mono- or diglycerides). Fatty acid, such as, but notlimited to, oleic acid and its glyceride derivatives, are useful in thepreparation of injectables, as are natural pharmaceutically acceptableoils, such as, but not limited to, olive oil or castor oil,polyoxyethylated versions thereof. These oil solutions or suspensionsalso can contain a long chain alcohol diluent or dispersant such as, butnot limited to, carboxymethyl cellulose, or similar dispersing agents.Other commonly used surfactants, such as, but not limited to, Tweens orSpans or other similar emulsifying agents or bioavailability enhancers,which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms also can be used for thepurpose of formulation.

A composition for oral administration can be any orally acceptabledosage form including capsules, tablets, emulsions and aqueoussuspensions, dispersions, and solutions. In the case of tablets,commonly used carriers include, but are not limited to, lactose and cornstarch. Lubricating agents, such as, but not limited to, magnesiumstearate, also are typically added. For oral administration in a capsuleform, useful diluents include, but are not limited to, lactose and driedcorn starch. When aqueous suspensions or emulsions are administeredorally, the active ingredient can be suspended or dissolved in an oilyphase combined with emulsifying or suspending agents. If desired,certain sweetening, flavoring, or coloring agents can be added.

Pharmaceutical compositions for topical administration according to thedescribed invention can be formulated as solutions, ointments, creams,suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils.Alternatively, topical formulations can be in the form of patches ordressings impregnated with active ingredient(s), which can optionallyinclude one or more excipients or diluents. In some preferredembodiments, the topical formulations include a material that wouldenhance absorption or penetration of the active agent(s) through theskin or other affected areas.

A topical composition contains a safe and effective amount of adermatologically acceptable carrier suitable for application to theskin. A “cosmetically acceptable” or “dermatologically-acceptable”composition or component refers a composition or component that issuitable for use in contact with human skin without undue toxicity,incompatibility, instability, or allergic response. The carrier enablesan active agent and optional component to be delivered to the skin at anappropriate concentration(s). The carrier thus can act as a diluent,dispersant, solvent, or the like to ensure that the active materials areapplied to and distributed evenly over the selected target at anappropriate concentration. The carrier can be solid, semi-solid, orliquid. The carrier can be in the form of a lotion, a cream, or a gel,in particular one that has a sufficient thickness or yield point toprevent the active materials from sedimenting. The carrier can be inertor possess dermatological benefits. It also should be physically andchemically compatible with the active components described herein, andshould not unduly impair stability, efficacy, or other use benefitsassociated with the composition.

Combination Therapies

In some embodiments, the pharmaceutical composition may further includean additional compound having antiproliferative activity. The additionalcompound having antiproliferative activity can be selected from a groupof antiproliferative agents including those shown in Table 12.

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be formulated and employed incombination therapies, that is, the compounds and pharmaceuticalcompositions can be formulated with or administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder, or they may achieve different effects(e.g., control of any adverse effects).

By “antiproliferative agent” is meant any antiproliferative agent,including those antiproliferative agents listed in Table 12, any ofwhich can be used in combination with a creatine transport and/orcreatine kinase inhibitor to treat the medical conditions recitedherein. Antiproliferative agents also include organo-platinederivatives, naphtoquinone and benzoquinone derivatives, chrysophanicacid and anthroquinone derivatives thereof.

TABLE 12 Alkylating agents Busulfan Chlorambucil dacarbazineprocarbazine ifosfamide altretamine hexamethylmelamine estramustinephosphate thiotepa mechlorethamine dacarbazine streptozocin lomustinetemozolomide cyclophosphamide Semustine Platinum agents spiroplatinlobaplatin (Aeterna) tetraplatin satraplatin (Johnson Matthey)ormaplatin BBR-3464 (Hoffmann-La Roche) iproplatin SM-11355 (Sumitomo)ZD-0473 (AnorMED) AP-5280 (Access) oxaliplatin cisplatin carboplatinAntimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision)2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC(Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabinegemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecanmesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposidegimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan(Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho)dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone(Novuspharma) J-107088 (Merck & Co) rebeccamycin analogue (Exelixis)BNP-1350 (BioNumerik) BBR-3576 (Novuspharma) CKD-602 (Chong Kun Dang)rubitecan (SuperGen) KW-2170 (Kyowa Hakko) irinotecan (CPT-11)hydroxycamptothecin (SN-38) topotecan Antitumor antibiotics valrubicinazonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazonelosoxantrone plicamycin MEN-10755 (Menarini) porfiromycin GPX-100 (GemPharmaceuticals) mitoxantrone (novantrone) Epirubicin amonafidemitoxantrone doxorubicin Antimitotic colchicine E7010 (Abbott) agentsvinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer)dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197(Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258(Aventis) combretastatin A4 (BMS) epothilone B (Novartis)isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126(AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (EliLilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma)auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS)BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797(BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075(GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin Apaclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi)atamestane (BioMedicines) formestane letrozole exemestane anastrazoleThymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthaseinhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagoniststrabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (BaxterInternational) mafosfamide (Baxter International) albumin + 32P (IsotopeSolutions) apaziquone (Spectrum thymectacin (NewBiotics)Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferasearglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitorslonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma)BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidartrihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate(Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer)pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (Aton Pharma)depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG)Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex)inhibitors marimastat (British Biotech) BMS-275291 (Celltech)Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis)reductase inhibitors triapine (Vion) didox (Molecules for Health) TNFalpha virulizin (Lorus Therapeutics) revimid (Celgene)agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott)YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoicacid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptoragonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy(Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK(Progenies) Technology) adenocarcinoma vaccine (Biomira) ISF-154(Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2(Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira)synchrovax vaccines (CTL Immuno) MGV (Progenies) melanoma vaccine (CTLImmuno) β-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy(Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCamBiotherapeutics) abatacept (BMS) MPDL3280A (Genentech) pembrolizumabMEDI4736 Hormonal and estrogens dexamethasone antihormonal agentsconjugated estrogens prednisone ethinyl estradiol methylprednisolonechlortrianisen prednisolone idenestrol aminoglutethimidehydroxyprogesterone caproate leuprolide medroxyprogesterone octreotidetestosterone mitotane testosterone propionate; P-04 (Novogen)fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosteronearzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofinebicalutamide goserelin flutamide Leuporelin nilutamide bicalutamidePhotodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda)agents Theralux (Theratechnologies) lutetium texaphyrin (Pharmacyclics)motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitorsimatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia)kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib(Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518(Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia)Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190(AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210(Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447(Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth)IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774(Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis)SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) EmodinGenistein Radicinol Radicinol Vemurafenib (B-Raf enzyme Met-MAb (Roche)inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo)ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMPagonist, Ribapharm) BCX-1777 (PNP inhibitor, BioCryst) alvocidib (CDKinhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247(COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor,Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent,SRI CapCell ™ (CYP450 stimulant, Bavarian Nordic) International) GCS-100(gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent,Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen(NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, AllosTherapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88(heparanase inhibitor, Progen) seocalcitol (vitamin D receptor agonist,Leo) tesmilifene (histamine antagonist, YM 131-I-TM-601 (DNA antagonist,BioSciences) TransMolecular) histamine (histamine H2 receptor agonist,Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDHinhibitor, Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide(integrin antagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist,Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinaseinhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE Vinhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst)CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer,AG-2037 (GART inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogenactivator inhibitor, Wilex) Immunol ™ (triclosan oral rinse, Endo)PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine(uridine prodrug, Wellstat) bortezomib (proteasome inhibitor,Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant,SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor,TransMID-107 ™ (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosispromotor, Procyon) PT-100 (growth factor agonist, Point doranidazole(apoptosis promotor, Pola) Therapeutics) cafestol Chrysophanic acidkahweol Cesium oxides caffeic acid BRAF inhibitors, Tyrphostin AG PDL1inhibitors PD-1 inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumabsorafenib angiogenesis inhibitors BRAF inhibitors rituximab (CD20antibody, Genentech urocidin (apoptosis promotor, Bioniche) carmustineRo-31-7453 (apoptosis promotor, La Roche) Mitoxantrone brostallicin(apoptosis promotor, Pharmacia) Bleomycin β-lapachone Absinthin gelonindabrafenib CRS-207 midostaurin (PKC inhibitor, Novartis) CHS-828(cytotoxic agent, Leo) bryostatin-1 (PKC stimulant, GPC Biotech)trans-retinoic acid (differentiator, NIH) CDA-II (apoptosis promoter,Everlife) MX6 (apoptosis promoter, MAXIA) SDX-101 (apoptosis promoter,Salmedix) apomine (apoptosis promoter, ILEX Oncology)

The invention features the following numbered embodiments:

1. A compound having the structure:

wherein X¹ is absent, NH, or CH₂;

R¹ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R², R³, and R⁴ are independently hydrogen or optionally substitutedC₁-C₆ alkyl; and

R⁵ and R⁶ are hydrogen or NH₂;

wherein if R⁵ and R⁶ are both hydrogen or R⁵ is NH₂ and R⁶ is hydrogenthen R² is optionally substituted C₁-C₆ alkyl,

or a pharmaceutically acceptable salt thereof.

2. The compound of embodiment 1, wherein R¹ is hydrogen.

3. The compound of embodiments 1 or 2, wherein R³ and R⁴ are hydrogen.

4. The compound of any one of embodiments 1-3, wherein R² is hydrogen oroptionally substituted C₁-C₆ alkyl, wherein said optionally substitutedC₁-C₆ alkyl is methyl, ethyl, isopropyl, propyl, isobutyl, or optionallysubstituted C₁-C₆ haloalkyl.

5. The compound of embodiment 4, wherein said optionally substitutedC₁-C₆ haloalkyl is trifluoromethyl.

6. The compound of any one of embodiments 1-5, wherein R⁵ and R⁶ areboth hydrogen and R² is optionally substituted C₁-C₆ alkyl.

7. The compound of embodiment 6, wherein said optionally substitutedC₁-C₆ alkyl is methyl, ethyl, isopropyl, or isobutyl.

8. The compound of any one of embodiments 1-5, wherein R⁵ and R⁶ areboth NH₂.

9. The compound of embodiment 8, wherein R² is hydrogen.

10. The compound of embodiment 8, wherein R² is optionally substitutedC₁-C₆ alkyl.

11. The compound of embodiment 10, wherein said optionally substitutedC₁-C₆ alkyl is methyl or isopropyl.

12. The compound of any one of embodiments 1-5, wherein R⁵ is NH₂, R⁶ ishydrogen, and R² is optionally substituted C₁-C₆ alkyl.

13. The compound of embodiment 12, wherein said optionally substitutedC₁-C₆ alkyl is methyl or isopropyl.

14. The compound of any one of embodiments 1-5, wherein R⁵ is hydrogenand R⁶ is NH₂.

15. The compound of embodiment 14, wherein R² is hydrogen.

16. The compound of embodiment 14, wherein R² is optionally substitutedC₁-C₆ alkyl.

17. The compound of embodiment 16, wherein said optionally substitutedC₁-C₆ alkyl is methyl or isopropyl.

18. The compound of any one of embodiments 1-17, wherein X¹ is absent.

19. The compound of any one of embodiments 1-17, wherein X¹ is CH₂.

20. The compound of any one of embodiments 1-17, wherein X¹ is NH₂.

21. The compound of embodiment 1, wherein said compound is any one ofthe compounds of Tables 1-3.

22. A compound having the structure:

wherein Q¹ is optionally substituted amidino or optionally substituted2-pyridyl;

X² is S or NR¹²;

m is 0 or 1;

R⁷ is hydrogen, optionally substituted C₁-C₆ alkyl, or optionallysubstituted C₆-C₁₀ aryl C₁-C₆ alkyl;

R⁸ and R⁹ are independently hydrogen, deuterium, halo, hydroxyl, N H₂,optionally substituted C₁-C₆ alkyl, or R⁸ or R⁹ can combine with R¹⁰ orR¹¹ to form an optionally substituted C₃-C₆ cycloalkyl ring or with R¹²to form an optionally substituted C₃-C₆ heterocycle;

R¹⁰ and R¹¹ are independently hydrogen, deuterium, optionallysubstituted C₁-C₆ alkyl, or R¹⁰ or R¹¹ can combine with R⁸ or R⁹ to forman optionally substituted C₃-C₆ cycloalkyl ring;

R¹² is hydrogen, optionally substituted C₁-C₆ alkyl, or R¹² can combinewith R⁸ or R⁹ to form an optionally substituted C₃-C₆ heterocycle,

wherein if Q¹ is optionally substituted 2-pyridyl then R¹² is hydrogen,and

-   -   wherein if R⁹ is halo then R⁸ is halo or optionally substituted        C₁-C₆ alkyl,

or a pharmaceutically acceptable salt thereof.

23. The compound of embodiment 22, wherein R⁷ is hydrogen.

24. The compound of embodiments 22 or 23, wherein m is 1.

25. The compound of any one of embodiments 22-24, wherein R⁹ ishydrogen, deuterium, or halo.

26. The compound of embodiment 25, wherein said halo is fluoro.

27. The compound of any one of embodiments 22-26, wherein R¹¹ ishydrogen or deuterium.

28. The compound of any one of embodiments 22-27, wherein R⁸ and R¹⁰combine to form an optionally substituted C₃-C₆ cycloalkyl ring.

29. The compound of embodiment 28, wherein said optionally substitutedC₃-C₆ cycloalkyl ring is cyclopropyl or cyclobutyl.

30. The compound of any one of embodiments 22-27, wherein both R¹⁰ andR¹¹ are deuterium.

31. The compound of embodiment 30, wherein R⁸ and R⁹ are both deuterium.

32. The compound of any one of embodiments 22-27, wherein both R⁸ and R⁹are halo.

33. The compound of embodiment 32, wherein said halo is fluoro.

34. The compound of any one of embodiments 22-27, wherein R¹⁰ isoptionally substituted C₁-C₆ alkyl.

35. The compound of embodiment 34, wherein said optionally substitutedC₁-C₆ alkyl is optionally substituted C₁-C₆ haloalkyl.

36. The compound of embodiment 35, wherein said optionally substitutedC₁-C₆ haloalkyl is trifluoromethyl.

37. The compound of embodiment 36, wherein R⁸ is NH₂.

38. The compound of any one of embodiments 22-27, wherein R⁸ is NH₂.

39. The compound of embodiment 38, wherein R¹⁰ is optionally substitutedC₁-C₆ alkyl.

40. The compound of embodiment 39, wherein said optionally substitutedC₁-C₆ alkyl is methyl.

41. The compound of any one of embodiments 22-40, wherein Q¹ isoptionally substituted amidino.

42. The compound of embodiment 41, wherein said optionally substitutedamidino has the structure:

43. The compound of embodiments 41 or 42, wherein X² is NR¹².

44. The compound of embodiment 43, wherein R⁸ and R¹² combine to form anoptionally substituted C₃-C₆ heterocycle.

45. The compound of embodiment 44, wherein said optionally substitutedC₃-C₆ heterocycle is azetidine.

46. The compound of embodiment 43, wherein R¹² is hydrogen.

47. The compound of embodiments 41 or 42, wherein X² is S.

48. The compound of any one of embodiments 22-40, wherein Q¹ isoptionally substituted 2-pyridyl.

49. The compound of embodiment 48, wherein said optionally substituted2-pyridyl has the structure:

50. The compound of embodiments 48 or 49, wherein X² is NR¹² and R¹² ishydrogen.

51. The compound of embodiment 22, wherein said compound is any one ofthe compounds of Tables 4-6.

52. A compound having the structure:A-B  Formula III

wherein A is a inhibitor of creatine transport comprising an amidinogroup;

B has the structure:

wherein n is 0 or 1;

Q² is hydroxyl, optionally substituted amino, or —SO₂OH; and

R¹³ and R¹⁴ are independently hydrogen, —CO₂H, or combine to form C═O;

wherein B is conjugated to A at one of the amidino nitrogens,

or a pharmaceutically acceptable salt thereof.

53. The compound of embodiment 52, wherein R¹⁴ is hydrogen.

54. The compound of embodiment 53, wherein R¹³ is —CO₂H.

55. The compound of embodiment 53, wherein R¹³ is hydrogen.

56. The compound of embodiments 52, wherein R¹³ and R¹⁴ combine to formC═O.

57. The compound of any one of embodiments 52-56, wherein n is 0.

58. The compound of any one of embodiments 52-56, wherein n is 1.

59. The compound of any one of embodiments 52-58, wherein Q² isoptionally substituted amino.

60. The compound of embodiment 59, wherein said optionally substitutedamino is —NH₂ or

61. The compound of any one of embodiments 52-58, wherein Q² ishydroxyl.

62. The compound of any one of embodiments 52-58, wherein Q² is —SO₂OH.

63. The compound any one of embodiments 52-62, wherein said inhibitor ofcreatine transport has the structure of a compound of any one ofembodiments 1-51 or any one of the compounds of Table 7 or Table 8.

64. The compound of embodiment 52, wherein said compound is any one ofthe compounds of Table 9 or Table 10.

65. A method for treating cancer, comprising administering to a subjectin need thereof, a compound of any one of embodiments 1-64 in an amountsufficient to treat said cancer.

66. A method of slowing the spread of a migrating cancer, comprisingadministering to a subject in need thereof, a compound of any one ofembodiments 1-64 in an amount sufficient to slow the spread of saidmigrating cancer.

67. The method of embodiment 66, wherein said method comprises thesuppression of metastatic colonization of said migrating cancer in theliver.

68. The method of embodiment 67, wherein said migrating cancer ismetastatic cancer.

69. The method of embodiment 68, wherein the metastatic cancer comprisescells exhibiting migration and/or invasion of migrating cells.

70. The method of embodiments 68 or 69, wherein said metastatic cancercomprises cells exhibiting endothelial recruitment and/or angiogenesis.

71. The method of any one of embodiments 67-70, wherein said migratingcancer spreads via seeding the surface of the peritoneal, pleural,pericardial, or subarachnoid spaces.

72. The method of any one of embodiments 67-70, wherein said migratingcancer spreads via the lymphatic system.

73. The method of any one of embodiments 67-70, wherein said migratingcancer spreads hematogenously.

74. The method of any one of embodiments 67-70, wherein said migratingcancer is a cell migration cancer.

75. The method of embodiment 74, wherein said cell migration cancer is anon-metastatic cell migration cancer.

76. The method of embodiment 75, where said cell migration cancer isovarian cancer, mesothelioma, or primary lung cancer.

77. A method for inhibiting proliferation or growth of cancer stem cellsor cancer initiating cells, comprising contacting the cell with acompound of any one of embodiments 1-64 in an amount sufficient toinhibit proliferation or growth of said cell.

78. A method of reducing the rate of tumor seeding of a cancercomprising administering to a subject in need thereof a compound of anyone of embodiments 1-64 in an amount sufficient to reduce tumor seeding.

79. A method of reducing or treating metastatic nodule-forming of cancercomprising administering to a subject in need thereof a compound of anyone of embodiments 1-64 in an amount sufficient to treat said metastaticnodule-forming of cancer.

80. The method of any one of embodiments 65-79, wherein said cancer isbreast cancer, colon cancer, renal cell cancer, non-small cell lungcancer, hepatocellular carcinoma, gastric cancer, ovarian cancer,pancreatic cancer, esophageal cancer, prostate cancer, sarcoma, ormelanoma.

81. The method of any one of embodiments 65-79, wherein said cancer isgastrointestinal cancer.

82. The method of embodiment 81, wherein said gastrointestinal cancer isesophageal cancer, stomach cancer, pancreatic cancer, liver cancer,gallbladder cancer, colorectal cancer, anal cancer, mucosa-associatedlymphoid tissue cancer, gastrointestinal stromal tumors, a cancer of thebiliary tree, or a gastrointestinal carcioid tumor.

83. The method of any one of embodiments 65-82, wherein said cancer is adrug resistant cancer.

84. The method of any one of embodiments 65-83 further comprisingadministering an additional antiproliferative agent.

85. The method of embodiment 84, wherein said additionalantiproliferative agent is capecitabine, gemcitabine, fluorouracil,FOLFOX (5-FU, leucovorin, and Eloxatin), FOLFIRI (5-FU, leucovorin, andCamptosar), EOX (Epirubicin, Oxaliplatinum, and Xeloda), Taxotere,Erbitux, Zaltrap, Vectibix, Ramucirumab, Tivozanib, Stivarga, CRS-207,or a PD-1 or PDL-1 antibody.

86. A method of treating metastatic cancer in a subject in need thereofcomprising:

(a) providing a subject identified to have, or to be at risk of having,metastatic cancer on the basis of the expression level of miR-483-5pand/or miR-551a is below a predetermined reference value or theexpression level of CKB and/or SLC6a8 is above a predetermined referencevalue; and

(b) administering to said subject an effective amount of a compound ofany one of embodiments 1-64.

87. A method for treating metastatic cancer in a subject in needthereof, comprising contacting creatine transport channel SLC6a8 with acompound of any one of embodiments 1-64 in an amount effective tosuppress metastatic colonization of said cancer.

EXAMPLES

Materials and Methods

Cell Culture

Indicated cell-lines were purchased from ATCC and cultured in DMEM mediasupplemented with 10% FBS, sodium pyruvate, L-glutamine andpenicillin-streptomycin antibiotics. For drug pre-treatment, cells weretreated with indicated amounts of drug for 24-48 hrs.

Animal Studies

All animal work was conducted in accordance with a protocol approved bythe Institutional Animal Care and Use Committee (IACUC) at TheRockefeller University. 5-6 weeks old age-matched male NOD-SCID micewere used for intrahepatic colonization and liver metastasis assays.

Proliferation Assay in Hypoxic Conditions

100K cells were seeded in triplicates in 6 well plates and cells werecounted 5 days after seeding. Cells were cultured in cell culturechamber containing 1% oxygen.

Primary Tumor Growth

1×10⁶ cells were suspended in 100 μl of 1:1 PBS:Matrigel mixture andinjected into the subcutaneous flanks of mice. Tumor growth was measuredusing digital calipers starting 7 days after injection when palpabletumors can be measured accurately. Volume of the tumors were calculatedusing the formula, Volume=(width)²×(length)/2. When treated with drugs,mice were injected with indicated amounts of drug in 300 μl PBS dailyuntil the mice were euthanized.

Metastasis Assay

5×10⁶ highly metastatic cancer cells were injected into the portalcirculation of immunodeficient mice. One day after inoculation of cancercells, mice were injected indicated amounts of drug in 300 μL PBS.Treatment was continued daily and metastatic progression was monitoredby bioluminescent imaging until the mice were euthanized at which pointlivers were excised for bioluminescent imaging and gross histology.Where indicated, cancer cells were pre-treated with indicated amounts ofcompound for 48 hrs before injection into immunodeficient mice.

In Vivo Creatine Transporter Inhibition Assay

Soluble compounds were formulated in saline solution (0.9% NaCl). Somecompounds were first dissolved 1 N hydrochloric acid (1.0 equivalent) tomake the HCI salt followed by addition of PBS to adjust to the finalvolume. Less soluble compounds were first dissolved 1 or 2 Nhydrochloric acid (1.0 equivalent) to make the HCI salt followed byaddition of DMSO and water to adjust to the final volume resulting in a1:1 DMSO to aqueous ratio.

Studies were performed on 6-7 week old C57B16 male mice, receiving aregular diet (Purina 5001, Research Diet) and on a regular sleep rhythm(12h night/day schedule). Experiments were performed ˜6h after exposingto daylight. Mice were weighed and randomly divided into groups of 3mice per group and injected i.p. with 100-200 μL of dosing solution todeliver 250 mg/kg (50 mg/mL or 381 mM) β-GPA equivalent (i.e. 1.91mmol/kg) along with a vehicle control. Creatine-(methyl-d₃) monohydrate(i.e. creatine-d₃, Cambridge Isotope Laboratories, Catalog DLM-1302) wasdissolved in 100 μL 0.9% NaCl (0.2 mg/mL) and injected i.p. 7 minutesafter drug injection. Volumes were adjusted based on the weight of themice to reach a final dose of 1 mg/kg. After one hour, mice wereeuthanized, hearts were perfused with PBS, removed, snap-frozen inliquid nitrogen, and stored at −80° C. until further processing.

Mouse hearts were thawed and weighed into 1.5 mL Eppendorf conicaltubes. Typical heart weights range from 800-1200 mg. Between six totwelve 1 mm zirconia/silica beads (BioSpec Products, Inc., Bartlesville,Okla.) were added to the tubes with sufficient volume of 70% 2-propanolin water to afford a 4-fold dilution. The samples were then placed in aMiniBeadBeater (BioSpec Products, Inc.) for 2 minutes to disrupt thetissue and homogenize the sample.

Aliqouts (20 μL) of the homogenized hearts were transferred to a 96-wellmicrotiter plate. Samples were extracted by the addition of acetonitrile(1.0 mL) containing 0.25 μg/mL of creatine-d₅ (CDN Isotopes,Pointe-Claire, Quebec) as internal standard. Samples were mixed on arotary shaker for 10 minutes then placed in a centrifuge to spin for 10minutes at 3000 rpm at 4° C. Supernatant (900 μL) was transferred to96-well deep well plate for analysis.

Calibration standards, blanks, and quality control samples are preparedfrom control mouse hearts homogenized as noted above. Aliquots ofhomogenate were then spiked with known quantities of creatine-d₃ (CDNIsotopes, Pointe-Claire, Quebec) or solvent, and processed along withthe samples as noted above.

Analysis was conducted by LC-MS/MS using an Acquity UPLC (Waters Corp.,Milford, Mass.)/Triple Quad 5500 (AB Sciex, Framingham, Mass.) system.Five microliters of sample are injected onto a HILIC column, 2.1×50 mm,3 μm (Fortis Technologies, Cheshire, England) at a flow rate of 0.4mL/min. A binary gradient of acetonitrile and 10 mM ammonium acetate wasused to elute analytes from the column. The mass spectrometer wasoperated in positive ion electrospray in Multiple Reaction Monitoringmode for the following mass transitions:

Creatine-d₅: m/z 137.1/95.0

Creatine-d₃: m/z 135.1/93.0

Data were collected and processed using Analyst 1.6.2 (AB Sciex,Framingham, Mass.). A linear calibration of the creatine-d₃/creatine-d₅peak area ratio ranged from 0.05 to 10 μg/mL. Data was report as μg ofcreatine-d₃ per gram of heart. Mean values and standard deviations werecalculated from three heart samples and percent creatine-d₃ transportinhibition was reported relative to vehicle control.

TABLE 13 Percent Inhibition of Creatine-d₃ Transport in Heart Tissue.Compound % Inhibition of Creatine-d₃ Transport 219 79.0 (+/−1.2)  2201.4 (+/−8.2) 258 71.8 (+/−5.9)  261 24.7 (+/−12.6) 358 11.1 (+/−13.6)376 39.1 (+/−25.2) 125  4.5 (+/−10.6)  28 (β-GPA) 73.4 (+/−6.7) 

In Vivo Selection

1×10⁶ LS174T cells expressing a luciferase reporter were suspended in avolume of 20 μl 1:1 PBS/Matrigel mixture and injected intra-hepaticallyinto the livers of NOD-SCID mice. Metastatic nodules were allowed todevelop over a period of 3-4 weeks and monitored by bioluminescenceimaging. Nodules formed were excised and dissociated by collagenase andhyaluronidase digestion into single cell suspension. The cells wereallowed to expand in in vitro before re-injection into mice. After threere-iterations of in vivo selection, highly metastatic LvM3a and LvM3bderivative cell-lines were established.

Lenti-miR Library Screening

Cells were transduced with a lentivirus Lenti-miR library of 611 miRNAs(System Biosciences) at a low multiplicity of infection (MOI) such thateach cell over-expressed a single miRNA. The transduced population wasthen injected intra-hepatically into NOD-SCID mice for in vivo selectionof miRNAs that when over-expressed, either promoted or suppressedmetastatic liver colonization. Genomic DNA PCR amplication and recoveryof lenti-viral miRNA inserts were performed on cells prior to injectionand from liver nodules according to manufacturer's protocol. miRNA arrayprofiling allowed for miRNA insert quantification prior to and after invivo selection.

Organotypic Slice Culture System

Cells to be injected were labeled with cell-tracker red or green(Invitrogen) and inoculated into livers of NOD-SCID mice throughintrasplenic injection. The livers were then extracted and cut into 150um slices using a Mcllwain tissue chopper (Ted Pella) and plated ontoorganotypic tissue culture inserts (Millipore) and cultured in William'sE Medium supplemented with Hepatocyte Maintenance Supplement Pack(Invitrogen). After indicated time periods, the liver slices were fixedin paraformaldehyde and imaged using multi-photon microscopy.

In Vivo Caspase Activation Assay

To measure caspase activity in vivo, VivoGlo Caspase 3/7 Substrate(Z-DEVD-Aminoluciferin Sodium Salt, Promega) was used. The luciferin isinactive until the DEVD peptide is cleaved from by activated caspase-3in apoptotic cells. DEVD-luciferin was injected into mice bearingcolorectal cancer cells expressing luciferase. Upon activation byapoptotic cells, bioluminescence imaging can be performed to measurecaspase activity in vivo. Five hours after in vivo caspase activitymeasurement, mice are injected with regular luciferin for normalizationpurposes.

Adeno-Associated Viral Therapy

miR-483-5p and miR-551a were cloned as a polycistron consisting of bothmiRNA precursor with flanking genomic sequences in tandem into the BgIIIand NotI site of scAAV.GFP (Plasmid 21893, Addgene). Listed below aregenomic sequences encoding for miR-483-5p and miR-551a (SEQ ID NOs: 5and 6), corresponding precursor sequences (underlined, SEQ ID NOs: 3 and4), and corresponding mature microRNA sequences (underlined and in bold,SEQ ID NOs: 1 and 2). Adeno-associated viruses were packaged, purifiedand titered using the AAV-DJ Helper Free expression system from CellBiolabs.

miR-551a: GGAGAACCTTCAGCTTCATGTGACCCAGAGACTCCTGTATGCCTGGCTCTGGGAGTACAGAAGGGCCTAGAGCTGACCCCTGCCCTCCGAAGCCCCTGGGGCACTAGATGGATGTGTGCCAGAGGGTAGTAGAGGCCTGGGGGTAGAGCCCAGCACCCCCTTCGCGTAGAGACCTGGGGGACCAGCCAGCCCAGCAACCCCCTCGCGGCCGACGCCTGAGGCTGTTCCTGGCTGCTCCGGTGGCTGCCAGAGGGGACTGCCGGGTGACCCTGGAAATCCAGAGTGGGTGGGGCCAGTCTGACCGTTTCTAGGCGACCCACTCTTGGTTTCCAGGGTTGCCCTGGAAACCACAGATGGGGAGGGGTTGATGGCACCCAGCCTCCCCCAAGCCTGGGAAGGGACCCCGGATCCCCAGAGCCTTTCCCTGCCTATGGAGCGTTTCTCTTGGAGAACAGGGGGGCCTCTCAGCCCCTCAATGCAAGTTGCTGAG miR-483-5p:CCTGCCCCATTTGGGGGTAGGAAGTGGCACTGCAGGGCCTGGTGCCAGCCAGTCCTTGCCCAGGGAGAAGCTTCCCTGCACCAGGCTTTCCTGAGAGGAGGGGAGGGCCAAGCCCCCACTTGGGGGACCCCCGTGATGGGGCTCCTGCTCCCTCCTCCGGCTGATGGCACCTGCCCTTTGGCACCCCAAGGTGGAGCCCCCAGCGACCTTCCCCTTCCAGCTGAGCATTGCTGTGGGGGAGAGGGGGAAGACGGGAGGAAAGAAGGGAGTGGTTCCATCACGCCTCCTCACTCCTCTCCTCCCGTCTTCTCCTCTCCTGCCCTTGTCTCCCTGTCTCAGCAGCTCCAGGGGTGGTGTGGGCCCCTCCAGCCTCCTAGGTGGTGCCAGGCCAGAGTCCAAGCTCAGGGACAGCAGTCCCTCCTGTGGGGGCCCCTGAACTGGGCTCACATCCCACACATTTTCCAAACCACTCCCATTGTGAGCCTTTGGTCCTGGTGGTGTCCCTCTGGTTGTGGGACCAAGAGCTTGTGCCCATTTTTCATCTGAGGAA GGAGGCAGCListed below are the corresponding RNA sequencesfor SEQ ID NOs: 1-4 (SEQ ID NOs: 7-10) (SEQ ID NO: 7)GACCCACUCUUGGUUUCCA (SEQ ID NO: 8)GGGGACUGCCGGGUGACCCUGGAAAUCCAGAGUGGGUGGGGCCAGUCUGACCGUUUCUAGGCGACCCACUCUUGGUUUCCAGGGUUGCCCUGGAAA (SEQ ID NO: 9)GAAGACGGGAGGAAAGAAGGGAG (SEQ ID NO: 10)GAGGGGGAAGACGGGAGGAAAGAAGGGAGUGGUUCCAUCACGCCUCCUCACUCCUCUCCUCCCGUCUUCUCCUCUC

CKB, SLC6a8 Knockdown

pLKO vectors expressing shRNA hairpins targeting CKB and SLC6a8 wereordered from Sigma-Aldrich. Two independent hairpins that gave the bestknockdown of transcript levels were used for all experiments. Thesehairpin DNA and RNA sequences are listed below in Table 14:

TABLE 14 Selected hairpin DNA and RNA sequences SEQ ID SEQ ID NameDNA Sequences NO RNA Sequences NO CKB CCGGCCCAGATTGAAACT 11CCGGCCCAGAUUGAAACUC 15 CTCTTCACTCGAGTGAA UCUUCACUCGAGUGAAGAGGAGAGTTTCAATCTGGG AGUUUCAAUCUGGGUUUUU TTTTT CKB CCGGCCGCGGTATCTGG 12CCGGCCGCGGUAUCUGGC 16 CACAATGACTCGAGTCAT ACAAUGACUCGAGUCAUUGTGTGCCAGATACCGCGG UGCCAGAUACCGCGGUUUU TTTTTTG UUG shSLCCCGGGCTGGTCTACAAC 19 CCGGGCUGGUCUACAACAA 20 6a8 #2 AACACCTACTCGAGTAGGCACCUACUCGAGUAGGUGU TGTTGTTGTAGACCAGCT UGUUGUAGACCAGCUUUUU TTTTG G shSLCCCGGCTTATTCCCTACGT 13 CCGGCUUAUUCCCUACGUC 17 6a8 #4 CCTGATCCTCGAGGATCACUGAUCCUCGAGGAUCAGG GGACGTAGGGAATAAGTT ACGUAGGGAAUAAGUUUUU TTTG G shSLCCCGGATTACCTGGTCAAG 14 CCGGAUUACCUGGUCAAGU 18 6a8 #5 TCCTTTACTCGAGTAAAGCCUUUACUCGAGUAAAGGA GACTTGACCAGGTAATTT CUUGACCAGGUAAUUUUUU TTTG G

The following primers were used for quantitative qRT-PCR of SLC6a8:Forward Primer: 5′-GGC AGC TAC AAC CGC TTC AAC A-3′ and Reverse Primer:5′-CAG GAT GGA GAA GAC CAC GAA G-3′ (SEQ ID No. 21 and 22,respectively).

Cyclocreatine and Beta-Guanidiopropionic Acid Treatment

Mice were treated with 10 mg of cyclocreatine or saline vehicle,administered through intra-peritoneal injection. The treatment regimestarted one day after inoculation of tumor cells and continued until themice were euthanized. Beta-guanidipropionic acid was administered at adose of 200 μL of 0.5M solution through intra-peritoneal injection.Treatment regime were as that for cyclocreatine treatment.

Example 1. Synthesis of Creatine Transport Inhibitors and/or CreatineKinase Inhibitors of the Invention

Compounds of the invention may be synthesized using methods known in theart, for example using methods described in U.S. Pat. Nos. 5,321,030,5,324,731, 5,955,617, 5,994,577, or 5,998,457 or methods described inMetabolism 1980, 29 (7), 686, J. Med. Chem. 2001, 44, 1231, J. Biol.Chem. 1972, 247, 4382, J. Chem. Inf. Model. 2008, 48 (3), 556, or J.Med. Chem. 2001, 44, 1217. Alternatively, the compounds of the inventionmay be synthesized using the methods described below.

Abbreviations

-   ACN acetonitrile-   β-GPA 3-guanidinopropionic acid i.e. β-guanidinopropionic acid-   BINAP racemic 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   BLQ below level of quantification-   Boc tert-butyloxycarbonyl-   Br₂ bromine-   BrCN cyanogen bromide-   ° C. degrees Celcius-   ca. circa or approximately-   CAN ceric ammonium nitrate-   Cbz carbobenzyloxy-   CH₂Cl₂ dichloromethane-   Cul copper (I) iodide-   Cs₂CO₃ cesium carbonate-   D₂O deuterium oxide-   DCC dicyclohexylcarbodiimide-   DCI dicyclohexylcarbodiimide-   DCM dichloromethane or methylenechloride-   DIPEA diisopropylethylamine-   DMAP 4-dimethylaminopyridine or N,N-dimethylaminopyridine-   DME 1,2-dim ethoxyethane-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   eq. equivalents-   ES(pos)MS electrospray positive mode mass spectrometry-   EtOAC ethyl acetate-   EtOH ethanol-   Et₂O diethyl ether-   Fmoc fluorenylmethyloxycarbonyl chloride-   g gram(s)-   HPLC high performance liquid chromatography-   h hour-   H₂ hydrogen gas-   K₂CO₃ potassium carbonate-   K₃PO₄ potassium phosphate tribasic-   KOH potassium hydroxide-   LC/MS liquid chromatography mass spectrometry-   LC/MS/MS liquid chromatography tandem mass spectrometry-   LiOH lithium hydroxide-   M molar-   Mel methyl iodide-   MeOH methanol-   mg milligram(s)-   MgSO₄ magnesium sulfate-   min. minute(s)-   mL milliliters(s)-   mm millimeter(s)-   mmol millimole(s)-   MTS 2-methyl-2-thiopseudourea sulfate-   m/z mass to charge ratio-   N normal-   Na₂S₂O₃ sodium thiosulfate-   Na₂SO₄ sodium sulfate-   NaH sodium hydride-   NaHCO₃ sodium bicarbonate-   Nal sodium Iodide-   NalO₄ sodium periodate-   NaOCH₃ sodium methoxide-   NaOH sodium hydroxide-   NaNO₂ sodium nitrite-   NBS N-bromosuccinimide-   NMR nuclear magnetic resonance-   PhthNK potassium phthalimide-   Pd/C palladium on carbon-   Pd(OAc)₂ palladium (II) acetate-   PdCl₂ palladium (II) chloride-   psi pounds per square inch-   PyBOP benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium    hexafluorophosphate-   RuCl₃ ruthenium trichloride hydrate-   SO₂Cl₂ sulfuryl chloride-   SOCl₂ thionyl chloride-   TCl 1,1′-thiocarbonyldiimidazole-   TEA triethylamine-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   TLC thin layer chromatography-   TPP triphenylphosphine-   TSA p-toluenesulfonic acid-   General method to make cyclocreatine analogs of the invention

Cyclocreatine analogs may be made from amino carboxylic acids or estersand reaction with aziridines in inert solvents (e.g. diethyl ether, THF,DME, ethanol, DMF, THF, etc.). These diamine intermediates are thenreacted with cyanogen bromide in inert solvent (e.g. diethyl ether, THF,DMF, etc.) to form iminoimidazolidine desired products.

General Method to Make 1-carboxymethyl-2-iminohexahydropyrimidineAnalogs of the Invention

Iminohexahydropyrimidine analogs may be made from α-halo carboxylicacids or esters and reaction with 1,3-diaminopropanes. These diamineintermediates may be reacted with cyanogen bromide in inert solvent(e.g. diethyl ether, THF, DMF, etc.) to form iminohexahydropyrimidinedesired products.

General Method to Make Dihydrazinylimidazolidine andDihydrazinylhexahydropyrimidine Analogs of the Invention

Dihydrazinyl analogs may be made from α-halo carboxylic acids or estersand reaction with protected hydrazinyl alkyl amino derivatives.Protected hydrazinyl alkyl amino derivatives may be made by reaction oftrifluoroacetohydrazide with aziridines or 1-azido-3-halopropanefollowed by subsequent azide reduction under standard conditions (e.g.triphenylphosphine-acetone-water). These diamine intermediates may bereacted with 1,1′-thiocarbonyldiimidazole (TCl) in polar solvent (e.g.DMF, dioxane, etc.) to form imidazolidinethiones ortetrahydro-2-pyrimidinethiones. Activation with alkyl halide (e.g.methyl iodide) and subsequent reaction with hydrazine cleaves thetrifluoroacetamide protecting group and would afford the desiredproducts.

General Method to Make 2-hydrazinylimidazolidine and2-hydrazinylhexahydropyrimidine Analogs of the Invention

The diaminocarboxylic acid or ester intermediates generated above may bereacted with 1,1′-thiocarbonyldiimidazole (TCl) in polar solvent (e.g.DMF, dioxane, etc.) to form imidazolidinethiones ortetrahydro-2-pyrimidinethiones. Activation with alkyl halide (e.g.methyl iodide) and subsequent reaction with hydrazine would afford thedesired products.

General Method to Make Aminoiminoimidazolidine andAminoiminohexahydropyrimidine Analogs of the Invention

Aminoiminoimidazolidine and aminoiminohexahydropyrimidine analogs may bemade from α-halo carboxylic acids or esters and reaction with azirindesor azetidines respectively. These α-carboxy aziridines or azetidines areopened with cyanogen bromide to form cyanamide alkyl bromideintermediates (see J. Org. Chem. 1949, 14, 605 and J. Am. Chem. Soc.2013, 135(41), 15306). Reaction with hydrazine would afford the desiredcyclic products.

General Method to Make imino-1,2,4-triazinanes Analogs of the Invention

Imino-1,2,4-triazinanes analogs may be made from α-halo carboxylic acidsor esters and reaction with protected hydrazinyl alkyl azidoderivatives. PMB-protected hydrazinyl alkyl azide derivatives may beprepared as described in Org. Biomol. Chem. 2012, 10(30), 5811. Theazide is reduced under standard conditions (e.g.triphenylphosphine-acetone-water) and the resulting diamine is treatedwith cyanogen bromide to form the cyclic core. Deprotection of the PMBhydrazine protecting group under standard conditions (e.g. CAN, strongacid, etc.) would afford the desired product.

Synthesis of Compound 225

Alanine (2.0 mmol) is dissolved in diethyl ether (10 mL) and aziridine(2.0 mmol) is added. The mixture is heated to reflux for 2 h and cooledto room temperature. Cyanogen bromide (2.3 mmol) is added and thereaction is stirred at room temperature overnight. The precipitate isfiltered and washed with diethyl ether to afford2-(2-iminoimidazolidin-1-yl)propanoic acid (225).

Compounds 226-228 may be synthesized using similar methods as used tomake compound 225 by replacing alanine with α-aminobutanoic acid,valine, or isoleucine.

Synthesis of Compound 237

2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether and1,3-diaminopropane (2.0 mmol) is added. The reaction is stirredovernight at room temperature and the precipitate is filtered and washedwith diethyl ether to afford the HCl salt of2-[(3-aminopropyl)amino]propanoic acid. The salt is dissolved in waterand sodium carbonate (2.5 mmol) is added followed by cyanogen bromide(2.3 mmol). The reaction is stirred at room temperature overnight. Thereaction is quenched with trifluoroacetic acid and the mixture isconcentrated under reduced pressure. The residue is purified by reversephase chromatography (acetonitrile/water with 0.05% trifluoroaceticacid) and the product is collected and lyophilized to afford thetrifluoroacetate salt of 2-(2-imino-1,3-diazinan-1-yl)propanoic acid(237).

Compounds 238-240 may be synthesized using similar methods as used tomake compound 237 by replacing 2-chloropropanoic acid with2-chlorobutanoic acid, 2-chloro-3-methylbutanoic acid, or2-chloro-3-methylpentanoic acid.

Synthesis of Compound 229

Step 1:2-{[2-(trifluoroacetohydrazido)ethyl]amino}acetic acid (INT-1)

Trifluoroacetohydrazide (2.0 mmol) and aziridine (2.0 mmol) aredissolved in diethyl ether (5 mL) and stirred at room temperatureovernight. 2-Chloroacetic acid (2.0 mmol) is added and the mixturestirred 3 h at room temperature. The precipitate is filtered and washedwith diethyl ether to afford INT-1.

Step 2:2-[2-sulfanylidene-3-(trifluoroacetamido)imidazolidin-1-yl]aceticacid (INT-2)

Diamine INT-1 (1.5 mmol) is dissolved in DMF (5 mL) and reacted with1,1′-thiocarbonyldiimidazole (1.5 mmol) at room temperature for 3 h. Thereaction is poured into 0.1 N aqueous HCl, the aqueous layer isextracted with ethyl acetate, the organic layer is dried over sodiumsulfate, concentrated under reduced pressure, and purified by silica gelcolumn chromatography (10-90 methanol-dichloromethane) to afford INT-2.

Step 3:2-[3-amino-2-hydrazinylideneimidazolidin-1-yl]acetic acid (229)

INT-2 (1.0 mmol) is dissolved in ethanol (5 mL) and methyl iodide isadded (1.0 mmol). The mixture is stirred for 1 h at room temperature.Upon completion, hydrazine (5 mmol) is added and the mixture is heatedto reflux for 8 h. The mixture is concentrated to remove excesshydrazine, the residue is purified by reverse phase chromatography(acetonitrile/water with 0.05% trifluoroacetic acid), and the product iscollected and lyophilized to afford the trifluoroacetate salt of2-[3-amino-2-hydrazinylideneimidazolidin-1-yl]acetic acid (229).

Compounds 230-228 and 241-243 may be synthesized using similar methodsas used to make compound 229 by replacing chloroacetic acid with2-chloroacetic acid, 2-chloropropanoic acid, or2-chloro-3-methylbutanoic acid. To synthesize compounds 241-243,aziridine may be replaced with 1,3-dibromopropane.

Synthesis of Compound 232

2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether andaziridine (2.0 mmol) is added. The reaction is stirred overnight at roomtemperature and the precipitate is filtered and washed with diethylether to afford the HCl salt of 2-(aziridin-1-yl)propanoic acid. Thesalt is dissolved in water, sodium carbonate (2.5 mmol) is addedfollowed by cyanogen bromide (2.3 mmol), and the reaction is stirred atroom temperature overnight. The reaction is quenched withtrifluoroacetic acid and the mixture is concentrated under reducedpressure. The residue is purified by reverse phase chromatography(acetonitrile/water with 0.05% trifluoroacetic acid) and the product iscollected and lyophilized to afford the trifluoroacetate salt of2-(3-amino-2-iminoimidazolidin-1-yl)propanoic acid (232).

Compounds 233 and 244-246 may be synthesized using similar methods tomake compound 232 by replacing 2-chloropropanoic acid with2-chloroacetic acid, or 2-chloro-3-methylbutanoic acid. To synthesizecompounds 244-246, aziridine may be replaced with azetidine.

Synthesis of Compound 234

Step 1:2-(2-sulfanylideneimidazolidin-1-yl)acetic acid (INT-3)

Glycine (2 mmol) is dissolved in diethyl ether (10 mL) and aziridine (2mmol) is added. The mixture is heat is to reflux for 2 h and cooled toroom temperature. The mixture is concentrated under reduced pressure,dissolved in DMF (5 mL), and reacted with 1,1′-thiocarbonyldiimidazole(1.5 mmol) at room temperature for 3 h. The reaction is poured into 0.1N aqueous HCl, the aqueous layer is extracted with ethyl acetate, theorganic layer is dried over sodium sulfate, concentrated under reducedpressure, and purified by silica gel column chromatography (10-90methanol-dichloromethane) to afford INT-3.

Step 2:2-[2-hydrazinylideneimidazolidin-1-yl]acetic acid (234)

INT-3 (1.0 mmol) is dissolved in ethanol (5 mL) and methyl iodide isadded (1.0 mmol). The mixture is stirred for 1 h at room temperature.Upon completion, hydrazine (5 mmol) is added and the mixture is heatedto reflux for 8 h. The mixture is concentrated to room excess hydrazine,the residue is purified by reverse phase chromatography(acetonitrile/water with 0.05% trifluoroacetic acid), and the product iscollected and lyophilized to afford the trifluoroacetate salt of2-[2-hydrazinylideneimidazolidin-1-yl]acetic acid (234).

Compounds 235-336 may be synthesized using similar methods to makecompound 234 by replacing glycine with alanine or valine. Compounds247-349 may be synthesized using similar methods to make the diamineintermediate for compound 237 and then following the protocol to make234.

Synthesis of Compound 250

Step 1:1-(2-azidoethyl)-1-[(4-methoxyphenyl)methyl]hydrazine (INT-4)

Synthesis of PMB-protected hydrazinyl ethyl azide is prepared asdescribed in Org. Biomol. Chem. 2012, 10(30), 5811.

Step2:2-[2-(2-aminoethyl)-2-[(4-methoxyphenyl)methyl]hydrazine-1-yl]aceticacid (INT-5)

2-Chloropropionic acid (2.0 mmol) is dissolved in diethyl ether (5 mL)and INT-4 (2.0 mmol) is added. The reaction is stirred overnight at roomtemperature and the precipitate is filtered and washed with diethylether to afford the HCl salt. The salt is dissolved in water-acetone(1:10, 5 mL) and triphenylphosphine (TPP) is added. The mixture isheated to 50° C. for 14 hours.

Step 3:2-(3-imino-1,2,4-triazinan-2-yl)acetic acid (250)

INT-5 (1.0 mmol) is dissolved in water, sodium carbonate (2.5 mmol) isadded followed by cyanogen bromide (1.3 mmol), and the reaction isstirred at room temperature overnight. The reaction is quenched withtrifluoroacetic acid and the mixture is concentrated under reducedpressure. The residue is purified by reverse phase chromatography(acetonitrile/water with 0.05% trifluoroacetic acid) and the product iscollected and lyophilized to afford the trifluoroacetate salt of2-(3-imino-1,2,4-triazinan-2-yl)acetic acid (250).

Compounds 251-252 may be synthesized using similar methods to makecompound 250 by replacing 2-chloroacetic acid with 2-chloropropanoicacid or 2-chloro-3-methylbutanoic acid.

General Method to Make Guanidine Containing Compounds of the Invention

Guanidines are made from amines using standard guanylating agents (e.g.2-methyl-2-thiopseudourea sulfate [MTS], cyanamide,N,N-di-Boc-1H-pyrazole-1-carboxamide, etc.). A preferred method usingMTS is described in J. Med. Chem. 2001, 44, 1217, J. Chem. Soc. C 1971,238 and Tetrahedron Lett. 1996, 37, 2483. Briefly, amines are dissolvedor suspended in basic aqueous alcoholic solvent and reacted with2-methyl-2-thiopseudourea sulfate (MTS) for 24-72 h or longer andprecipitated products are isolated by filtration. If necessary, esterhydrolysis is done by treatment with hydroxide ion (e.g. LiOH, NaOH,KOH, etc) in aqueous alcoholic solvent or THF, to afford the desiredproduct.

In some case, guanidines are made from amines under anhydrous conditionsusing pyrazole-activated guanylating agents (e.g.1H-pyrazole-1-carboxamidine hydrochloride,3,5-dimethyl-1-pyrazolylformaminidium nitrate,N-Boc-1H-pyrazole-1-carboxamidine,N,N′-di-Boc-1H-pyrazole-1-carboxamidine,N-(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine, andN,N′-bis(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine). Methods forusing pyrazole-activated guanylating agents are reviewed in Eur. J. Org.Chem. 2002, 3909. Briefly, amines are used in excess or with a base(e.g. triethylamine, diisopropylethylamine, etc.) and are dissolved insolvent (e.g. DMF, acetonitrile, THF, methanol, dichloromethane, etc.).The reaction is stirred for 4-72 h and at room temperature but in somecases heating is required. When protected pyrazole-activated guanylatingagents are used (i.e. W=Boc or Cbz) products can be purified by normalphase silica gel column chromatography. If necessary, ester hydrolysisis done by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) inaqueous alcoholic solvent or THF, to provide the carboxylic acid.Finally, standard deprotection conditions are used to remove theguanidine protecting groups (i.e. TFA removal of Boc or hydrogenation ofCbz) to afford the desired product.

In other cases, amino alcohols are used with the method above. Afterguanylation, the alcohol is oxidized to the carboxylic acid using sodiummetaperiodate and ruthenium (III) chloride (catalytic) in acetonitrile,ethyl acetate and water (Org. Lett. 2008, 10, 5155). Finally, standarddeprotection conditions are used to remove the guanidine protectinggroups (i.e. TFA removal of Boc or hydrogenation of Cbz) to afford thedesired product.

General Method to Make 2-aminopyridine Containing Compounds of theInvention

Aminopyridines may be made utilizing cross-coupling methods as describedin J. Am. Chem. Soc. 2008, 130(20), 6586 and J. Org. Chem. 1996, 61(21),7240. Briefly, amines, 2-halopyridines, and sufficient base (e.g. cesiumcarbonate, potassium tert-butoxide, etc.) are dissolved or suspended inpolar solvent (e.g. DMF, dioxane), catalytic palladium (e.g. PdCl₂ orPd(OAc)₂) and phosphine ligand (e.g. BINAP) are then be added and thereaction is heated to greater than 80° C. for 4-6 h. If necessary, esterhydrolysis by treatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc)in aqueous alcoholic solvent or THF, affords the desired product.

Alternatively, Buchwald amide-cross-coupling methods are used togenerate desired compounds as described in see J. Am. Chem. Soc. 2002,124, 11684 and J. Am. Chem. Soc. 2001, 123, 7727. Briefly, amines areprotected as amides or carbamates (e.g. trifluoroacetamide, Boc, Cbz,etc.) and reacted with 2-bromopyridine in polar solvent (e.g. dioxane,DMF) with base (e.g. potassium phosphate tribasic, cesium carbonate,potassium tert-butoxide, etc.), racemic trans-1,2-diaminocyclohexaneligand, and catalytic copper (I) iodide. The solution is degassed for 5minutes by bubbling nitrogen gas directly into the solution and themixture is heated at greater than 95° C. for 6-12 hours. Amineprotecting groups are removed using standard conditions and, ifnecessary, ester hydrolysis by treatment with hydroxide ion (e.g. LiOH,NaOH, KOH, etc) in aqueous alcoholic solvent or THF, affords the desiredproduct.

General Method to Make Pseudothiourea Containing Compounds of theInvention

Pseudothioureas may be made by reaction of a thiourea with alkylhalidesas described in J. Med. Chem. 2001, 44, 1217. Briefly, 3-chloropropionicacids or esters may be reacted with thiourea in polar solvent (e.g.acetone) followed by refluxing the mixture for 48 h. 3-Chloropropionicacids or esters may be made by reaction of 3-hydroxypropionic acids withthionyl chloride as described in International Patent Publication No.WO9933785.

Alternatively, pseudothioureas may be made from amino compounds viaalkyl bromides using methods as described in Tetrahedron: Asymmetry1998, 9(10), 1641 and International Patent Publication No. WO2002009705.Briefly, 3-aminocarboxylic acids or esters may be converted to3-bromocarboxylic acids or esters via activation with sodium nitrite inthe presence of hydrobromic acid. The resulting bromo compound may bereacted with a thiourea in a suitable solvent (e.g. toluene or acetone)at greater than 60° C. for 4-24 h. If necessary, ester hydrolysis bytreatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueousalcoholic solvent, would afford the desired product.

General Method to Make 2-aminopseudothiourea Containing Compounds of theInvention

2-Aminopseudothioureas may be made from aziridines by methods similar tothose described in Chem. Comm. 2000, 7, 619 and Org. Biomol. Chem. 2005,3(18), 3357. Briefly, N-Cbz-2-carboxyaziridines may be reacted withthiourea and borontrifluoride-etherate in inert solvent (e.g.chloroform, dichloromethane, etc). Subsequent hydrogenation in thepresence of palladium on carbon, and if necessary, ester hydrolysis bytreatment with hydroxide ion (e.g. LiOH, NaOH, KOH, etc) in aqueousalcoholic solvent, would afford the desired product. 2-Carboxyaziridinesmay be made by methods as described in Synlett 2001, 5, 679 andTetrahedron Lett. 2006, 47(13), 2065 and may be converted toCbz-protected aziridines by standard methods.

Many starting materials for compounds of the invention are commerciallyavailable or methods for synthesis are known in the literature. Table 15lists starting materials for synthesis of compounds of the invention andprovides literature references for uncommon reagents.

TABLE 15 Starting materials for the synthesis of selected compounds #Structure Starting Material CAS Number Reference SM01

(3R)-3-aminobutyric acid 3775-73-3 Org. Process Res. Dev. 2011, 15, 1130SM02

(3S)-3-aminobutyric acid 3775-72-2 Org. Process Res. Dev. 2011, 15, 1130SM03

β-alanine-2,2,3,3-D₄ 116173-67-2 J. Labelled Compd. Ra. 1988, 25(2), 217SM04

β-alanine-3,3-D₂ 116173-66-1 J. Labelled Compd. Ra. 1988, 25(2), 217SM05

2,2-difluoro-3-amino- propanoic acid 428452-49-7 Tetrahedron Lett. 2003,44(11), 2375; PCT Int. Appl., 2007062308 SM06

3-azetidinecarboxylic acid 36476-78-5 Commercially available SM07

3-amino-4,4,4- trifluorobutanoic acids 584-20-3 Commercially availableSM08

tert-butyl (1R,2S)-2- aminocyclopropane-1- carboxylate 150626-49-6 JP05155827 A 19930622 SM09

tert-butyl (1S,2R)-2- aminocyclopropane-1- carboxylate 150737-97-6 JP05155827 A 19930622; International Patent Publication No. WO 2008123207SM10

ethyl (1R,2R)-2-{[(tert- butoxy)carbonyl]amino} cyclopropane-1-carboxylate 613261-17-9 J. Org. Chem. 2003, 65(20), 7884; Org. Lett.2013, 75(4), 772; SM11

ethyl (1R,2R)-2- {[(benzyloxy)carbonyl] amino}cyclopropane-1-carboxylate 613261-16-8 J. Org. Chem. 2003, 68(20), 7884; Org. Lett.2013, 15(4), 772; in addition protocols as described in Tetrahedron2012, 68(47), 9566 can also be used to generate useful startingmaterials SM12

ethyl (1S,2S)-2- [(methoxycarbonyl) amino]cyclopropane-1- carboxylate1356459-72-7 J. Org. Chem. 2003, 66(20), 7884; Org. Lett. 2013, 15(4),772; in addition protocols as described in Tetrahedron 2012, 66(47),9566 can also be used to generate useful starting materials SM13

(1R,2S)-2- aminocyclobutane-1- carboxylic acid 221158-95-8 J. Org. Chem.2009, 74(8), 3217; J. Org. Chem. 2005, 70(20), 7963; Tetrahedron:Asymmetry 2000, 11(17), 3569 SM14

(1S,2R)-2- aminocyclobutane-1- carboxylic acid 648433-09-4 J. Org. Chem.2009, 74(8), 3217; J. Org. Chem. 2005, 70(20), 7963 SM15

(1R,2R)-2- aminocyclobutane-1- carboxylic acid 951173-26-5 J. Org. Chem.2009, 74(8), 3217 SM16

(1S,2S)-2- aminocyclobutane-1- carboxylic acid 951173-27-6 J. Org. Chem.2009, 74(8), 3217 SM17

2-methyl-3- azetidinecarboxylic acid 1638771-37-5 Chiral trans isomers:Synthesis 2005, 20, 3508; Chiral cis isomers: J. Org. Chem. 2012,77(17), 7212 SM18

2-hydroxy-3- azetidinecarboxylic acid 70807-37-3 Commercially available;International Patent Publication No. WO2011043817 SM19

2-amino-3- azetidinecarboxylic acid 138650-25-6 Commercially availableSM20

3-fluoro-3- azetidinecarboxylic acid 1363380-85-1 International PatentPublication No. WO2013019561; J. Org. Chem. 2009, 74(5), 2250; or madeby deprotection of 1-[(tert-butoxy)carbonyl]-3-fluoroazetidine-3-carboxylic acid [1126650-67-6] SM21

2-methyl-3- azetidinecarboxylic acid 1213240-07-3 Commerciallyavailable; or made by deprotection of 1-[(tert-butoxy)carbonyl]-3-methylazetidine-3- carboxylic acid [887591-62-0] SM22

(3S)-3-amino-4,4,4- trifluorobutanoic acids 151871-99-7 Chem. Comm.2012, 45(34), 4124 SM23

(3R)-3-amino-4,4,4- trifluorobutanoic acids 151911-19-2 Chem. Comm.2012, 45(34), 4124 SM24

(2S,3S)-2,3-diamino- 4,4,4-trifluorobutanoic acid 1632315-15-1 J.Fluorine Chem. 2015, 171, 67 SM25

ethyl (2S,3R)-2,3- diamino-4,4,4- trifluorobutanoate 1219366-64-9International Patent Publication No. WO2010031750 SM26

(2R,3R)-2,3-diamino- 4,4,4-trifluorobutanoic acid NA Made by a similarprotocol as [1632315-15-1] above but using (R)-N-[(1E)-ethylidene]-2-methylpropane-2- sulfinamide [1219607-85-8] SM27

ethyl (2R,3S)-2,3- diamino-4,4,4- trifluorobutanoate NA Made by asimilar protocol as [1219366-64-9] above but using (R)-N-[(1E)-ethylidene]-2-methylpropane-2- sulfinamide [1219607-85-8] SM28

(3R)-3-amino(4,4,4- ²H₃)butanoic acid NA Made according to proceduresdescribed for the synthesis of chiral 3- aminobutyric acid in Helv.Chim. Acta 1988, 71, 1824 and Tetrahedron: Asymmetry 1991, 3, 183, butreplacing crotonic acid with 2-butenoic-4,4,4-d₃ acid [1375453-29-4]made according to J. Magn. Reson. 2011, 270(1), 107 SM29

(3S)-3-amino(4,4,4- ²H₃)butanoic acid NA As above SM30

(S)-3-amino-4,4- difluoro-butanoic acid 111218-68-9 TetrahedronAsymmetry 1994, 5(6), 1119 SM31

(R)-3-amino-4,4- difluoro-butanoic acid 109537-89-5 TetrahedronAsymmetry 1994, 5(6), 1119 SM32

3-amino-4- fluorobutanoic acid 77162-47-1 Syn. Comm. 1985, 15(5), 377SM33

(3S)-3-aminopent-4- enoic acid 1389348-84-8 Made by hydrogenation ofethyl (3S)-3- aminopent-4-ynoate [149251-15-0] at 1 atm using Lindlar'scatalyst and then ester hydrolysis SM34

(3R)-3-aminopent-4- enoic acid 1388637-32-8 Made by hydrogenation ofethyl (3R)-3- aminopent-4-ynoate [188853-28-3] at 1 atm using Lindlar'scatalyst and then ester hydrolysis SM35

ethyl (3S)-3- aminopent-4-ynoate 149251-15-0 Bioorg. Med. Chem. Lett.1997, 7(13), 1699; U.S. Pat. No. 5,536,869; the ester can be hydrolyzedbefore or after the guanylation step SM36

ethyl (3R)-3- aminopent-4-ynoate 188853-28-3 Bioorg. Med. Chem. 1999,7(10), 2221; the ester can be hydrolyzed before or after the guanylationstep SM37

3-amino-pentanoic acid 18664-78-3 Commercially available SM38

(R)-3-amino-pentanoic acid 131347-76-7 Commercially available SM39

(S)-3-aminopentanoic acid 14389-77-6 Commercially available SM40

(3R)-3-aminohexanoic acid 775551-50-3 Synthesis 2008, 7, 1153 & Chem.Commun. 2007, 8, 849 SM41

(3S)-3-aminohexanoic acid 91298-66-7 ChemBioChem 2009, 10(9), 1558 &Synlett 1994, 10, 795 SM42

3-amino-4- methylpentanoic acid 5699-54-7 Commercially available SM43

(S)-3-amino-4- methylpentanoic acid 40469-85-0 Commercially availableSM44

(R)-3-amino-4- methylpentanoic acid 75992-50-6 Commercially availableSM45

3-amino-2,2- dimethylpropan-1-ol 26734-09-8 Commercially available;requires oxidation of the alcohol after guanylation SM46

[1- (aminomethyl) cyclopropyl]methanol 45434-02-4 Commerciallyavailable; requires oxidation of the alcohol after guanylation SM47

ethyl 1- (aminomethyl) cyclobutane-1- carboxylate 911060-83-8Commercially available; the ester can be hydrolyzed before or after theguanylation step SM48

3-amino-3- methylbutanoic acid 625-05-8 Commercially available SM49

2-(1- aminocyclopropyl) acetic acid 133616-20-3 Synlett 1991, 2, 87 SM50

2-(1-{[(tert- butoxy)carbonyl]amino} cyclobutyl)acetic acid 249762-02-5Commercially available; requires hydrolysis of the Boc protecting groupprior to guanylation SM51

(2R,3R)-3-amino-2- methylbutanoic acid 139344-67-5 U.S. Pat. Appl.Publ., 20110218342; Tetrahedron, 2007, 63(26), 5820 SM52

(2S,3R)-3-amino-2- methylbutanoic acid 863115-43-9 Made by an analogousprotocol described in Heterocycles 1999, 50(2), 677 for [39801-26-8] butusing (R)-(−)- N-methoxy-2-pyrrolidine carboxamide SM53

(2R,3S)-3-amino-2- methylbutanoic acid 39801-26-8 Heterocycles 1999,50(2), 677; J. Org. Chem. 1993, 55(8), 2282 SM54

(2S,3S)-3-amino-2- methylbutanoic acid 139344-68-6 J. Am. Chem. Soc.2005, 127(32), 11252; J. Org. Chem. 1993, 58(8), 2282 SM55

pyrrolidine-3- carboxylic acid 59378-87-9 Commercially available SM56

(3S)-pyrrolidine-3- carboxylic acid 72580-53-1 Commercially availableSM57

(3R)-pyrrolidine-3- carboxylic acid 72580-54-2 Commercially availableSM58

2-[(2S)-1-[(tert- butoxy)carbonyl] azetidin-2-yl]acetic acid1289384-58-2 Made from (S)-(tert- butoxycarbonyl)azetidine-2-carboxylicacid according to the protocol described in International PatentPublication No. WO2011111875; requires hydrolysis of the Boc protectinggroup prior to guanylation SM59

2-[(2R)-1-[(tert- butoxy)carbonyl] azetidin-2-yl]acetic acid1369534-61-1 Made from (R)-(tert- butoxycarbonyl)azetidine-2-carboxylicacid according to the protocol described in International PatentPublication No. WO2011111875; requires hydrolysis of the Boc protectinggroup prior to guanylation SM60

cis-3- aminocyclobutane-1- carboxylic acid 74316-27-1 Commerciallyavailable SM61

trans-3- aminocyclobutane-1- carboxylic acid 74307-75-8 Commerciallyavailable SM62

3-{[(tert- butoxy)carbonyl]amino}- 1- hydroxycyclobutane-1- carboxylicacid 1067239-17-1 International Patent Publication No. WO2011044538 &WO2008124821; requires hydrolysis of the Boc protecting group prior toguanylation SM63

ethyl 3-amino-1-{[(tert- butoxy)carbonyl]amino} cyclobutane-1-carboxylate NA ethyl 1-{[(tert-butoxy)carbonyl]amino}-3-hydroxycyclobutane-1-carboxylate [413597-67-8] (U.S. Pat. Appl. Publ.,20060292073) is converted to the amine by activation of the hydroxylgroup (e.g. tosyl chloride and pyridine), displacement with lithiumazide, and reduction to the amine (i.e. catalytic hydrogenation of Pd/Cor with triphenylphosphine) SM64

2-(azetidin-3-yl)acetic acid 183062-92-2 Commercially available; or madeby deprotection of 2-{1-[(tert- butoxy)carbonyl]azetidin-3-yl}aceticacid [183062-96-6] prior to guanylation SM65

L-(2S)-2,3- diaminopropionic acid 4033-39-0 Commercially available SM66

β-chloroalanine 51887-88-8 (D) 13215-35-5 (D/L) Org. Biomol. Chem. 2005,3(18), 3357 SM67

(2S,3R)-2-amino-3- chlorobutanoic acid 64233-79-0 Make desiredstereoisomers from L- threonine and L-allo-threonine using a similarprocedure described in: International Patent Publication No. WO9933785SM68

3-chloropropionic- 2,2,3,3-d₄ acid 1219802-17-1 Commercially availableSM69

ethyl 3-bromo-2,2- difluoropropionate 111773-24-1 Commercially availableSM70

ethyl 3-chloro-4,4,4- trifluorobutyrate 1309602-63-8 Commerciallyavailable SM71

(2S,3R)-2,3- diaminobutanoic acid 25023-80-7 Org. Biomol. Chem. 2003,1(21), 3708; Synlett 1996, 7, 621; Tetrahedron 2001, 57(39), 8267 SM72

(2S,3S)-2,3- diaminobutanoic acid 80999-51-5 Org. Biomol. Chem. 2003,1(21), 3708; Synlett 1996, 7, 621; Tetrahedron 2001, 57(39), 8267 SM73

2,3-diaminopropionic acid 54897-59-5 Commercially availableSynthesis of Compound 219

(R)-Aminobutyric acid SM01 (750 mg, 7.27 mmol) and2-methyl-2-thiopseudourea sulfate (MTS, 1.21 g, 4.36 mmol) weresuspended in methanol (4 mL). After addition of 3N sodium hydroxide inwater (2.62 mL, 1.09 eq.), the clear solution was stirred for 3 days atroom temperature. Subsequently, the white precipitate was filtered offand washed with water/methanol (15 mL, ½). The white powder was airdried for 1 hour and then put under high vacuum for 2 days to yield the(3R)-3-carbamimidamidobutanoic acid (219) as a white solid (879 mg,83%); ¹H-NMR (300 MHz, D₂O): δ 3.81 (m, 1H), 2.31 (m, 2H), 1.15 (d, 3H);ES(pos)MS m/z 146.1 (M+H⁺).

Synthesis of Compound 220

(S)-Aminobutyric acid SM02 (750 mg, 7.27 mmol) was converted into thecorresponding (3S)-3-carbamimidamidobutanoic acid (220) according to theprocedure for compound 219. The desired product was obtained as a whitepowder after high vacuum drying for 3 days (756 mg, 72%); ¹H-NMR (300MHz, D₂O): δ 3.84 (m, 1H), 2.35 (m, 2H), 1.18 (d, 3H). ES(pos) MS m/z145.9 (M+H⁺).

Synthesis of Compound 221

A suspension of (2S,3R)-2,3-diaminobutanoic acid SM71 (25 mmol) and2-methyl-2-thiopseudourea sulfate (MTS, 25 mmol) in methanol (25 mL) isstirred for 5 days at room temperature under a nitrogen atmosphere. Thereaction is then cooled to 0° C., filtered through a medium porosityglass frit, and the collected solid is washed with water and dried toprovide compound 221 as a mixture of regioisomers. The material ispurified by preparative HPLC chromatography and the product is collectedand lyophilized to afford compound 221.

Synthesis of Compound 223

2-chloro-pyridine (25 mmol), palladium (II) acetate (2.5 mmol), racemic2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (2.5 mmol) and cesiumcarbonate (65 mmol) are dissolved in toluene (75 mL) in a previouslydegassed sealed vessel. The mixture is flushed with nitrogen gas.Methyl-(R)-3-aminobutyrate SM01 (20 mmol) is added to the solution undernitrogen and the sealed mixture is heated overnight at 100° C. Thereaction is cooled to room temperature, diluted with diethyl ether andwashed with pH 7 buffer and water. The organic layer is concentrated andpurified by silica gel column chromatography (10:90methanol-dichloromethane) to afford (3R)-3-[(pyridin-2-yl)amino]butanoicacid (223).

Synthesis of Compound 257

Step 1: (1H-1,2,3-benzotriazol-1-ylmethyl)dibenzylamine (INT-6)

Hydroxymethylbenztriazol (10.4 g, 69.7 mmol) and dibenzylamine (13.4 mL,69.7 mmol) were converted to INT-6 (95% yield) according to the protocolin US2009/054414.

Step 2:ethyl 3-(dibenzylamino)-2,2-difluoropropanoate (INT-7)

Ethyl bromodifluoro acetate (4.8 g, 23.65 mmol) and INT-6 (7.78 g, 23.7mmol) were coupled according to the protocol in US2009/054414 to affordINT-7 (53% yield).

Step 3:ethyl 3-amino-2,2-difluoropropanoate TFA salt (INT-8)

The INT-7 (1.52 g, 4.59 mmol) was dissolved in ethanol (25 mL).Trifluoroacetic acid (0.372 mL, 4.85 mL) and a catalytic amount ofpalladium hydroxide on carbon was added and the reaction was subjectedto hydrogen under atmospheric pressure for 16 h. Subsequently, thecatalyst was removed by filteration through celite and washed withethanol. The resulting filtrate was evaporated under reduced pressureand the residue was treated with toluene (50 mL) and concentrated, thiswas repeated twice to remove residual solvent and excess water. Theresidue was dried under high vacuum overnight to afford the INT-8 as ayellowish oil (used as crude in the next step).

Step 4:ethyl3-{[(14-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-2,2-difluoropropanoate(INT-9)

The crude TFA salt of INT-8 was dissolved in dry THF (10 mL) and thentreated with triethylamine (1.36 mL, 9.73 mmol) andN,N′-di-Boc-1H-pyrazole-1-carboxamidine (1.57 g, 5.05 mmol). Afterstirring overnight at room temperature, the reaction mixture was pouredinto ethyl acetate (200 mL) and then washed with water (2×100 mL)adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. Thecombined aqueous washes were then re-extracted with ethyl acetate (100mL). The second organic phase was in turn washed with acidified water(100 mL) using 1N HCl to adjust the to pH 1-2. The combined organicphases were dried over magnesium sulfate, filtered, evaporated, andpurified by silica gel column chromatography to afford INT-9 as aviscous oil that solidified upon standing (1.25 g, 70% for two steps);¹H-NMR (300 MHz, DMSO-d₆): δ 11.42 (s, 1H). 8.60 (t, 1H), 4.26 (q, 2H),4.05 (td, 2H), 1.49 (s, 9H), 1.39 (s, 9H), 1.26 (t, 3H).

Step 5:Compound 257

INT-9 (0.5 g) is dissolved in THF (5 mL) and 1N LiOH is added (2.5 mL).Once ester hydrolysis is complete the mixture is concentrated undervacuum and the residue is dissolved in 1:4 trifluoroaceticacid-dichloromethane (5 mL). The mixture is stirred at room temperatureovernight to remove the Boc-protecting groups. The mixture isconcentrated under vacuum, the residue is purified by preparative HPLCchromatography, and the product is collected and lyophilized to affordcompound 257. Alternatively, the ester in INT-8 is hydrolyzed usinglithium hydroxide to make SM05 and the resulting amino acid isguanylated using 1H-pyrazole-1-carboxamidine hydrochloride anddiisopropylethylamine in DMF as shown below for compound 261.

Synthesis of Compound 258

3-Azetidinecarboxylic acid SM06 (2.0 g, 19.8 mmol) was suspended inwater (2 mL) and then treated with 10 N sodium hydroxide solution inwater (1.96 mL, 19.6 mmol). The thick yellowish solution was thentreated with solid cyanamide (1.01 g, 24 mmol) and the mixture turnedsolid instantly. More water (5 mL) was added to ensure adequate stirringand the slurry was then stirred at room temperature for 3 days. Thewhite solid was subsequently filtered off, washed with cold water (10mL, 0° C.) and then air dried for 2 hours. High vacuum drying for 2 daysaffords compound 258 as a white solid (1.48 g, 52%); ¹H-NMR (300 MHz,D₂O): δ 4.19 (t, 2H), 4.07 (dd, 2H), 3.34 (m, 1H); ES(pos) MS m/z 143.9(M+H⁺).

Synthesis of Compound 261

3-Amino-4,4,4-trifluorobutyric acid SM07 (500 mg, 3.18 mmol) anddiisopropylethylamine (1.16 mL, 6.68 mmol) were dissolved in dry DMF (3mL). After addition of 1H-pyrazole-1-carboxamidine hydrochloride (593mg, 3.50 mmol), the reaction mixture was stirred for 20 days at roomtemperature. The precipitate was filtered off, washed withmethanol/water (2/1, 15 mL) and air-dried. High vacuum drying overnightaffords the desired compound 261 as a white powder (135 mg, 21%); ¹H-NMR(300 MHz, D₂O): δ 4.09 (m, 1H), 2.47 (dd, 1H), 2.22 (dd, 1H); ES(pos) MSm/z 200.07 (M+H⁺).

Synthesis of Compound 275

Thiourea (100 mmol) and β-chloro-D-alanine hydrochloride SM66 (100 mmol)in acetone (22 mL) is stirred at reflux for 48 h. Acetone (ca. 150 mL)is added, and the mixture is stirred vigorously to promotesolidification. The solid is broken up, stirred until fine, filteredunder nitrogen and washed with acetone. The crude solid is dissolved inwarm 2-propanol (120 mL) and diluted with diethyl ether until cloudy (80mL) and crystallization to afford(2R)-2-amino-3-(carbamimidoylsulfanyl)propanoic acid (275).

Synthesis of Compound 278

To a flask is added cis-2-am inocyclobutane-1-carboxylic acid (18.8mmol) and 24.5 mL of 5 N hydrobromic acid. The reaction is cooled in anice bath to 0-5° C., followed by drop-wise addition of sodium nitrite(30.1 mmol) in 7.5 mL of water over five hours. The temperature ismaintained below 5° C. during the addition. After the addition iscomplete, the reaction is stirred for 12 hours at room temperature. Thereaction is diluted with diethyl ether (15 mL), the aqueous layer isremoved and the organic phase is washed with 1 N hydrochloric acid (15mL). The combined aqueous layers are washed with ethyl acetate (10 mL)and the combined organic extracts are dried of magnesium sulfate,filtered and concentrated under reduced pressure. The solid isrecrystallized from ethyl acetate (10 mL) and hexanes (10 mL) to obtaintrans-2-bromocyclobutane-1-carboxylic acid.

To a suspension of thiourea (15 mmol) in toluene (50 mL) in an oil bathat 60° C. is added trans-2-bromocyclobutane-1-carboxylic acid (2.5mmol). The reaction is stirred at 60° C. for 5 h. The toluene is thenremoved under reduced pressure and the resulting residue is diluted withwater (25 mL) and 1 N hydrochloric acid (30 mL), to achieve pH of 1-1.5.The mixture is stirred at room temperature for 1-2 hours and thenextracted with ethyl acetate (3×50 mL). The combined organic layers aredried over magnesium sulfate, filtered and concentrated under reducedpressure. The resulting solid is dissolved in water (3.0 mL) andfiltered through a 0.2 μm nylon filter. The filtrate is purified bypreparative HPLC (e.g. Waters PrepPak cartridge Delta-Pak C18compression column, 15 μm 25×100 mm, 95:5 water-acetonitrile at 12.0 mL/m in). The product is collected and lyophilized to afford the productcis-2-(carbamimidoylsulfanyl)cyclopropane-1-carboxylic acid (278).

Synthesis of Compound 286

Step 1:1-Benzyl 2-methyl 3-(trifluoromethyl)aziridine-1,2-dicarboxylate(INT-10)

INT-10 may be made by methods as described in Synlett 2001, 5, 679 andTetrahedron Lett. 2006, 47(13), 2065 and converted to a Cbz-protectedaziridine by standard methods (see Org. Biomol. Chem. 2005, 3(18),3357).

Step 2:methyl2-{[(benzyloxy)carbonyl]amino}-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoate(INT-11)

INT-10 (1.0 mmol) is dissolved in dichloromethane (10 mL) and themixture is cooled to 0° C. Borontrifluoride-etherate (1.0 mmol, 1 Mdichloromethane) is added drop-wise and the reaction is warmed to roomtemperature. The reaction is poured into 0.1 N aqueous HCl, the aqueouslayer is extracted with ethyl acetate, the organic layer is dried oversodium sulfate, concentrated under reduced pressure, and purified bysilica gel column chromatography (10-90 methanol-dichloromethane) toafford INT-11.

Step 3:2-amino-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoic acid(286)

INT-11 (0.5 mmol) is dissolved methanol, 10% palladium on carbon (Pd/C10 mol %) is added and the flask is vacuum purged with hydrogen gas 5times. The mixture is stirred vigorously at room temperature under ahydrogen atmosphere for 16 h. Nitrogen gas is used to purge the flaskand the mixture is filtered through Celite to remove the Pd/C. Themixture is concentrated under reduced pressure, and the residue istreated with 1N sodium hydroxide in methanol. Once ester hydrolysis iscomplete, the mixture is concentrated again under reduced pressure, andthe resulting residue is dissolved in water (3.0 mL) and filteredthrough a 0.2 μm nylon filter. The filtrate is purified by preparativeHPLC (e.g. Waters PrepPak cartridge Delta-Pak C18 compression column, 15μm 25×100 mm, 95:5 water-acetonitrile at 12.0 mL/min). The product iscollected and lyophilized to afford the product2-amino-3-(carbamimidoylsulfanyl)-4,4,4-trifluorobutanoic acid (286).

Synthesis of Compound 358

Am inopentanoic acid SM37 (500 mg, 4.27 mmol) was converted into thecorresponding guanidino derivative according to the procedure forcompound (219). The desired compound 358 was obtained as a white powderafter high vacuum drying (291 mg, 43%); ¹H-NMR (300 MHz, D₂O): δ 3.63(m, 1H), 2.39 (dd, 1H), 2.29 (dd, 1H), 1.52 (m, 2H), 0.85 (t, 3H);ES(pos) MS m/z 160.11 (M+H⁺).

Synthesis of Compound 376

Pyrrolidine-3-carboxylic acid hydrochloride (500 mg, 3.30 mmol) wasconverted into the desired derivative (376) according to a modifiedprocedure described for compound 258 where the amount of base wasincreased to neutralize the HCl-salt of the starting material. The finalmaterial was isolated as a white powder (198 mg, 38%); ¹H-NMR (300 MHz,D₂O): δ 3.4 (m, 4H), 2.99 (m, 1H), 2.17 (m, 1H), 2.03 (m, 1H); ES(pos)MS m/z 158.09 (M+H⁺).

Synthesis of Compound 366

Step 1:tert-butylN-[(1Z)-{[(tert-butoxy)carbonyl]imino}[(3-hydroxy-2,2-dimethylpropyl)amino]methyl]carbamate(INT-12)

3-Amino-2,2-dimethylpropanol SM45 (464 mg, 4.5 mmol) was dissolved indry THF (7 mL) and then treated withN,N′-di-Boc-1H-pyrazole-1-carboxamidine (1.24 g, 4.0 mmol). Afterstirring for 72 hours at room temperature, the reaction mixture waspoured into ethyl acetate (200 mL) and then washed with water (2×100 mL)adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. Thecombined aqueous washes were then re-extracted with ethyl acetate (100mL). The second organic phase was in turn washed with acidified water(100 mL) using 1N HCl to adjust the to pH 1-2. The combined organicphases were dried over magnesium sulfate, filtered, evaporated, andpurified by silica gel column chromatography to afford INT-12 as aviscous oil that solidified upon standing (1.32 g, 96%); ¹H-NMR (300MHz, DMSO-d₆): δ 11.48 (s, 1H). 8.55 (t, 1H), 4.93 (t, 1H), 3.18 (d,2H), 3.13 (d, 2H), 1.48 (s, 9H), 1.39 (s, 9H), 0.82 (s, 6H).

Step 2:3-{[(1Z)-{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}-2,2-dimethylpropanoicacid (INT-13)

INT-12 (1.30 g, 3.76 mmol) was dissolved in acetonitrile (25 mL), carbontetrachloride (25 mL), and water (40 mL). Sodium periodate (4.83 g, 22.6mmol) and ruthenium trichloride (50 mg, catalytic) were added and themixture was stirred for 3 h at room temperature or until TLC showedstarting material had been consumed. The resulting biphasic mixture waspoured into ethyl acetate (200 mL) and then washed with water (2×100 mL)adjusting the pH of the aqueous layer to pH 1-2 using 1N HCl. Thecombined aqueous washes were then re-extracted with ethyl acetate (100mL). The second organic phase was in turn washed with acidified water(100 m L) using 1N HCl to adjust the to pH 1-2. The combined organicphases were dried over magnesium sulfate, filtered, evaporated, andpurified by silica gel column chromatography to afford INT-13 as a solidfoam (1.05 g, 78%); ¹H-NMR (300 MHz, DMSO-d₆): δ 11.47 (s, 1H). 8.53 (t,1H), 3.42 (d, 2H), 1.47 (s, 9H), 1.39 (s, 9H), 1.13 (s, 6H).

Step 3:Compound 366

INT-14 is dissolved in 1:4 trifluoroacetic acid-dichloromethane (5 mL).The mixture is stirred at room temperature overnight to remove theBoc-protecting groups. The mixture is concentrated under vacuum and theresidue is purified by preparative HPLC chromatography to affordcompound 366.

In addition to the compounds described above, similar protocols are usedto make numerous analogs shown in Table 16 from starting materialslisted in Table 15.

TABLE 16 Starting materials for the synthesis of selected compoundsCompound # Table # Starting Material Protocol 253 4 SM08 or SM09 See 261254 4 SM10-SM12 See 261 255 4 SM03 See 219 256 4 SM04 See 219 257 4 SM05See 257 258 4 SM06 See 258 259 4 SM13 or SM14 See 219 260 4 SM15 or SM16See 219 261 4 SM07 See 261 262 4 SM24-SM27 See 261 327 4 SM08 See 261328 4 SM09 See 261 329 4 SM10 or SM11 See 261 330 4 SM12 See 261 331 4SM13 See 219 332 4 SM14 See 219 333 4 SM15 See 219 334 4 SM16 See 219335 4 SM17 See 258 336 4 SM17 See 258 337 4 SM17 See 258 338 4 SM17 See258 339 4 SM18 See 258 340 4 SM19 See 258 341 4 SM20 See 258 342 4 SM21See 258 343 4 SM22 See 261 344 4 SM23 See 261 345 4 SM24 See 261 346 4SM25 See 261 347 4 SM26 See 261 348 4 SM27 See 261 349 4 SM28 See 219350 4 SM29 See 219 351 4 SM30 See 261 352 4 SM31 See 261 353 4 SM32 See219 354 4 SM33 See 219 355 4 SM34 See 219 356 4 SM35 See 219 357 4 SM36See 219 358 4 SM37 See 358 359 4 SM38 See 219 360 4 SM39 See 219 361 4SM40 See 219 362 4 SM41 See 219 363 4 SM42 See 261 364 4 SM43 See 261365 4 SM44 See 261 366 4 SM45 See 366 367 4 SM46 See 366 368 4 SM47 See261 369 4 SM48 See 261 370 4 SM49 See 261 371 4 SM50 See 261 372 4 SM51See 219 373 4 SM52 See 219 374 4 SM53 See 219 375 4 SM54 See 219 376 4SM55 See 376 377 4 SM56 See 376 378 4 SM57 See 376 379 4 SM59 See 258380 4 SM58 See 258 381 4 SM60 See 219 382 4 SM61 See 219 383 4 SM62 See219 384 4 SM63 See 219 385 4 SM64 See 258 263 5 SM65 See 223 264 5 SM72See 223 265 5 SM71 See 223 266 5 SM08 or SM09 See 223 267 5 SM10-SM12See 223 268 5 SM03 See 223 269 5 SM04 See 223 270 5 SM05 See 223 271 5SM13 or SM14 See 223 272 5 SM15 or SM16 See 223 273 5 SM07 See 223 274 5SM24-SM27 See 223 386 5 SM73 See 223 387 5 SM71 See 223 388 5 SM72 See223 389 5 SM08 See 223 390 5 SM09 See 223 391 5 SM10 or SM11 See 223 3925 SM12 See 223 393 5 SM13 See 223 394 5 SM14 See 223 395 5 SM15 See 223396 5 SM16 See 223 397 5 SM17 See 223 398 5 SM17 See 223 399 5 SM17 See223 400 5 SM17 See 223 401 5 SM18 See 223 402 5 SM19 See 223 403 5 SM20See 223 404 5 SM21 See 223 405 5 SM22 See 223 406 5 SM23 See 223 407 5SM24 See 223 408 5 SM25 See 223 409 5 SM26 See 223 410 5 SM27 See 223411 5 SM28 See 223 412 5 SM29 See 223 413 5 SM30 See 223 414 5 SM31 See223 415 5 SM32 See 223 416 5 SM33 See 223 417 5 SM34 See 223 418 5 SM35See 223 419 5 SM36 See 223 420 5 SM38 See 223 421 5 SM39 See 223 422 5SM45 See 223 423 5 SM46 See 223 424 5 SM47 See 223 425 5 SM48 See 223426 5 SM49 See 223 427 5 SM50 See 223 428 5 SM51 See 223 429 5 SM52 See223 430 5 SM53 See 223 431 5 SM54 See 223 432 5 SM58 See 223 433 5 SM59See 223 434 5 SM60 See 223 435 5 SM61 See 223 436 5 SM62 See 223 275 6SM66 See 275 276 6 SM67 See 275 277 6 SM67 See 275 278 6 SM08 or SM09See 278 279 6 SM10-SM12 See 278 280 6 SM03 See 278 281 6 SM68 See 275282 6 SM69 See 275 283 6 SM13 or SM14 See 275 284 6 SM15 or SM16 See 278285 6 SM07 See 278 286 6 SM24-SM27 See 286Generic Scheme to Make Sulfanyl N-amidinoprodrugs

Sulfanyl N-amidino prodrugs may be synthesized in several steps fromhomodisulfides via N-alkylthiol-phthalimide. Disulfides are commerciallyavailable or easily prepared from sulfhydryls using standard conditions(e.g. iodine-water U.S. Pat. No. 6,025,488, sodium iodide-peroxideSynthesis, 2007, 3286-3289, etc.). In step 1, disulfides are reactedwith bromine or sulfuryl chloride to generate sulfenyl bromides orsulfenyl chlorides in situ at −15° C. to 0° C. in inert solvent (e.g.dichloromethane, 1,2-dichloroethane, chloroform, etc.; see J. Org. Chem.1971, 36, 3828; and J. Org. Chem. 1986, 51 (26), 5333). The resultingmixture is then agitated for 5 to 30 minutes and subsequentlytransferred drop-wise to a suspension of potassium phthalimide in asuitable solvent such as 1,2-dichloroethane at −15° C. to 0° C. (see J.Med. Chem. 2009, 52 (14), 4142 and Bioorg. Med. Chem. Lett. 2007, 17,6629). In step 2, N-alkylthiol-phthalimides are reacted with amidino orguanidine compounds to afford the desired products (see J. Med. Chem.2009, 52 (14), 4142 and International Patent Publication No.WO2010100337).

Synthesis of Compound 287

Step 1:(2S)-2-amino-3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)sulfanyl]propanoicacid (INT-15)

L-cystine (5 mmol), phthalimide (5 mmmol), pyridine (10 mmol) aredissolved in acetonitrile (10 mL). Bromine (5 mmol) is added drop-wiseto the solution at 0° C. and the mixture is warmed to room temperatureand stirred overnight. The reaction is concentrated under reducedpressure and the residue is then purified by reverse phasechromatography (acetonitrile/water with 0.05% trifluoroacetic acid). Theproduct is collected and lyophilized to afford the trifluoroacetate saltof INT-15.

Step 2:(2S)-2-amino-3-{[{amino[(2-carboxyethyl)amino]methylidene}amino]sulfanyl}propanoicacid (287)

β-Guanidinopropionic acid (1.00 mmol), INT-15 (1.15 mmol) and potassiumcarbonate (1.15 mmol) are dissolved in anhydrous acetonitrile (10 ml)and stirred overnight. The reaction is concentrated under reducedpressure and the residue is then purified by reverse phasechromatography (acetonitrile/water with 0.05% trifluoroacetic acid). Theproduct is collected and lyophilized to afford the trifluoroacetate saltof 287.

In addition to the synthesis of compound 287 described above, additionalprodrugs may be made by combining different guanidine compounds (e.g.β-guanidinopropionic acid, β-guanidinobutanoic acid,2-(2-iminoimidazolidin-1-yl)acetic acid, L-homocystine, etc.) withdifferent activated thiols (e.g. derived from L-cystine,N,N-diacetyl-L-cystine, 2-(carboxymethyldisulfanyl)acetic acid, etc.) aslisted in Table 17.

TABLE 17 Starting materials for the synthesis of selected compounds Com-Disulfide Starting pound # Guanidine Compound Material [CAS] 288β-guanidinopropionic acid N,N′-diacetyl-L-cystine [5545-17-5] 289β-guanidinopropionic acid 2-(carboxymethyldisulfanyl)acetic acid[505-73-7] 290 β-guanidinopropionic acid 2,2′-Dithiobis(ethylammonium)sulphate [16214-16-7] 291 β-guanidinopropionic acid L-homocystine[626-72-2] 292 β-guanidinopropionic acid 2,2′-dithiodiethanesulfonicacid [45127-11-5] 293 β-guanidinobutanoic acid L-cystine [56-89-3] 294β-guanidinobutanoic acid N,N′-diacetyl-L-cystine [5545-17-5] 295β-guanidinobutanoic acid 2-(carboxymethyldisulfanyl)acetic acid[505-73-7] 296 β-guanidinobutanoic acid 2,2′-dithiobis(ethylammonium)sulphate [16214-16-7] 297 β-guanidinobutanoic acid L-homocystine[626-72-2] 298 β-guanidinobutanoic acid 2,2′-dithiodiethanesulfonic acid[45127-11-5] 299 2-(2-iminoimidazolidin-1- L-cystine [56-89-3] yl)aceticacid 300 2-(2-iminoimidazolidin-1- N,N′-diacetyl-L-cystine yl)aceticacid [5545-17-5] 301 2-(2-iminoimidazolidin-1-2-(carboxymethyldisulfanyl)acetic yl)acetic acid acid [505-73-7] 3022-(2-iminoimidazolidin-1- 2,2′-dithiobis(ethylammonium) yl)acetic acidsulphate [16214-16-7] 303 2-(2-iminoimidazolidin-1- L-homocystine[626-72-2] yl)acetic acid 304 2-(2-iminoimidazolidin-1-2,2′-dithiodiethanesulfonic yl)acetic acid acid [45127-11-5] 3052-(2-imino-1,3-diazinan-1- L-cystine [56-89-3] yl)acetic acid 3062-(2-imino-1,3-diazinan-1- N,N′-diacetyl-L-cystine yl)acetic acid[5545-17-5] 307 2-(2-imino-1,3-diazinan-1-2-(carboxymethyldisulfanyl)acetic yl)acetic acid acid [505-73-7] 3082-(2-imino-1,3-diazinan-1- 2,2′-dithiobis(ethylammonium) yl)acetic acidsulphate [16214-16-7] 309 2-(2-imino-1,3-diazinan-1- L-homocystine[626-72-2] yl)acetic acid 310 2-(2-imino-1,3-diazinan-1-2,2′-dithiodiethanesulfonic yl)acetic acid acid [45127-11-5] 3112-(1-methylguanidino)ace- L-cystine [56-89-3] tic acid 3122-(1-methylguanidino)ace- N,N′-diacetyl-L-cystine tic acid [5545-17-5]313 2-(1-methylguanidino)ace- 2-(carboxymethyldisulfanyl)acetic tic acidacid [505-73-7] 314 2-(1-methylguanidino)ace-2,2′-dithiobis(ethylammonium) tic acid sulphate [16214-16-7] 3152-(1-methylguanidino)ace- L-homocystine [626-72-2] tic acid 3162-(1-methylguanidino)ace- 2,2′-dithiodiethanesulfonic tic acid acid[45127-11-5] 317 2-(1,3-dimethylguani- L-cystine [56-89-3] dino)aceticacid 318 2-(1,3-dimethylguani- N,N′-diacetyl-L-cystine dino)acetic acid[5545-17-5] 319 2-(1,3-dimethylguani- 2-(carboxymethyldisulfanyl)aceticdino)acetic acid acid [505-73-7] 320 2-(1,3-dimethylguani-2,2′-dithiobis(ethylammonium) dino)acetic acid sulphate [16214-16-7] 3212-(1,3-dimethylguani- L-homocystine [626-72-2] dino)acetic acid 3222-(1,3-dimethylguani- 2,2′-dithiodiethanesulfonic acid dino)acetic acid[45127-11-5]Synthesis of Compound 323

Step 1:N-benzyl-2-oxoazetidine-1-carbothioamide

Azetidinone (5 mmol) is dissolved in tetrahydrofuran and the mixture iscooled in an ice bath. Sodium hydride (60% mineral oil dispersion, 6mmol) is added followed by benzyl thioisocyanate (5 mmol) and themixture is stirred to room temperature over 2 h. The reaction is pouredinto water, the aqueous layer is extracted with ethyl acetate, theorganic layer is dried over sodium sulfate, concentrated under reducedpressure and purified by silica gel column chromatography (50-50 ethylacetate-hexanes) to afford N-benzyl-2-oxoazetidine-1-carbothioamide.

Step 2:N,N′-dibenzyl-2-oxoazetidine-1-carboximidamide

To a stirring solution of N-benzyl-2-oxoazetidine-1-carbothioamide (2.5mmol) in methanol (5 mL) is added methyl iodide (7.0 mmol). The solutionis stirred for 2 h, and solvent is removed under reduced pressure. Theresidue is partitioned between ethyl acetate (15 mL) and saturatedaqueous bicarbonate solution (15 mL), the mixture is shaken and theorganic layer is dried over sodium sulfate, concentrated under reducedpressure and purified by silica gel column chromatography (50-50 ethylacetate-hexanes) to afford the intermediate pseudothiourea.

To a stirring solution of this pseudothiourea (1 mmol) in methanol (3mL) is added benzylamine (1 mmol). The resulting solution is heated toreflux for 12 h in a sealed tube, cooled to ambient temperature, themixture is concentrated under reduced pressure and purified by silicagel column chromatography (50-50 ethyl acetate-hexanes) to affordN,N′-dibenzyl-2-oxoazetidine-1-carboximidamide.

Step 3:2-oxoazetidine-1-carboximidamide

N,N′-Dibenzyl-2-oxoazetidine-1-carboximidamide (0.5 mmol) is dissolvedmethanol, 10% palladium on carbon (Pd/C 10 mol %) is added and the flaskis vacuum purged with hydrogen gas 5 times. The mixture is stirredvigorously at room temperature under a hydrogen atmosphere for 16 h.Nitrogen gas is used to purge the flask and the mixture is filteredthrough Celite to remove the Pd/C. 1N Hydrochloric acid in methanol isadded to precipitate the HCl salt of compound 323.

Synthesis of Compound 324

2-chloro-pyridine (25 mmol), palladium (II) acetate (2.5 mmol), racemic2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (2.5 mmol) and cesiumcarbonate (65 mmol) is dissolved in toluene (75 mL) in a previouslydegassed sealed vessel. The mixture is flushed with nitrogen gas.Azetidinone (20 mmol) is added to the solution under nitrogen and thesealed mixture is heated overnight at 100° C. The reaction is cooled toroom temperature, diluted with diethyl ether, and washed with saturatedbicarbonate solution and water. The organic layer is concentrated andpurified by silica gel column chromatography (2:98methanol-dichloromethane) to afford 1-(pyridin-2-yl)azetidin-2-one.

Synthesis of Compound 325

Step1:3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoicacid (INT-16)

β-alanine (1.0 mmol) and N,N-di-(Boc)-1H-pyrazole-1-carboxamide (1.0mmol) are suspended in pyridine (2.0 mL) and stirred at 25° C. for 2days. The homogenous reaction is treated with 1N NaOH and extracted intoethyl acetate. The aqueous layer is acidified (pH 3) with 1N HCl andthen extracted into ethyl acetate. The combined organic layers arewashed with brine, dried over sodium sulfate, filtered and concentratedunder reduced pressure to provide3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoicacid (INT-16).

Step 2:(2R,3R,4R,5R)-4-[(3-{[{[(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoyl)oxy]-5-(5-fluoro-2-oxo-4-{[(pentyloxy)carbonyl]amino}-1,2-Dihydropyrimidin-1-yl)-2-methyloxolan-3-yl-3-{[{(tert-butoxy)carbonyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]amino}propanoate(INT-17)

INT-17 (0.7 mmol) andbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (PyBOP) (0.6 mmol) are added to a solution ofcapecitabine (0.30 mmol) in dichloromethane (2.0 mL). The resultingmixture is stirred at 25° C. overnight. The dichloromethane is removedunder reduced pressure and the residue is taken up in ethyl acetate. Theorganic layer is washed with water, brine, dried over magnesium sulfatefiltered, and concentrated under reduced pressure. The residue ispurified by silica gel column chromatography (2:98methanol-dichloromethane) to afford the Boc-protected coupled compound(INT-17).

Step 3:(2R,3R,4R,5R)-4-[(3-carbamimidamidopropanoyl)oxy]-5-(5-fluoro-2-oxo-4-{[(pentyloxy)carbonyl]amino}-1,2-dihydropyrimidin-1-yl)-2-methyloxolan-3-yl3-carbamimidamidopropanoate (325)

Boc-Deprotection is accomplished by standard condition using neattrifluoroacetic acid, mixtures of trifluoroacetic acid (TFA) withmethylene chloride, or hydrochloride acid (HCl) in dioxane. The materialfrom the previous step INT-17 (0.20 mmol) is treated with 4N HCl/dioxane(2.0 mmol). The mixture is stirred at 25° C. overnight, concentratedunder reduced pressure, and then subjected to reverse phasechromatography (acetonitrile/water with 0.05% trifluoroacetic acid) toafford the desired product 325.

Synthesis of Compound 326

Step1:1-(benzyloxy)-3-[(3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]propan-2-yl-3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoate(INT-18)

INT-17 (2.2 mmol) andbenzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (PyBOP) (2.1 mmol) are added to a solution of3-(benzyloxy)propane-1,2-diol (1.0 mmol) in dichloromethane (10 mL). Theresulting mixture is stirred at 25° C. overnight. The dichloromethane isremoved under reduced pressure and the residue is taken up in ethylacetate. The organic layer is washed with water, brine, dried overmagnesium sulfate filtered, and concentrated under reduced pressure. Theresidue is purified by silica gel column chromatography (50:50 ethylacetate-hexane) to afford the desired compound INT-18.

Step2:1-[(3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]-3-[(chlorocarbonyl)oxy]propan-2-yl-3-{[bis({[(tert-butoxy)carbonyl]amino})methylidene]amino}propanoate(INT-19)

INT-18 (0.5 mmol) is dissolved methanol, 10% palladium on carbon (10%Pd/C, 0.05 mmol) is added, and the flask is vacuum purged with hydrogengas 5 times. The mixture is stirred vigorously at room temperature undera hydrogen atmosphere for 16 h. Nitrogen gas is used to purge the flaskand the mixture is filtered through Celite to remove the Pd/C and thesolvent is concentrated under reduced pressure. The residue is used asis in the next reaction.

A mixture of triphosgene (0.25 mmol), sodium carbonate (0.5 mmol), anddimethylformamide (0.018 mmol) as a catalyst, in toluene (2 mL) iscooled to 0° C. and stirred at this temperature for 30 min. A solutionof the residue from the previous reaction in toluene (2 mL) is addedslowly over a period 30 min. The reaction mixture is stirred at 0° C.for 8 h. The solid sodium carbonate is removed by filtration and thesolvent is concentrated under reduced pressure. The resulting residueINT-19 is used as-is in the next reaction.

Step3:1-[({1-[(4R,6R,6aS)-2,2,6-trimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]-5-fluoro-2-oxo-1,2,3,4-tetrahydropyrimidin-4-yl}carbamoyl)oxy]-3-[(3-{[bis({[tert-butoxy)carbonyl]amino})methylidene]amino}propanoyl)oxy]propan-2-yl-3-{[bis({[tert-butoxy)carbonyl]amino})methylidene]amino}propanoate(INT-20)

5-Fluoro-2′,3′-o-isopropylidenecytidine (5-FIPC) is made according toprotocols described in International Patent Publication Nos.WO2008144980 and WO2008131062. 5-FIPC (0.25 mmol) and pyridine (0.5mmol) are dissolved in dichloromethane (5 mL) and INT-12 is added as asolution in dichloromethane (5 mL). The mixture is stirred at roomtemperature for 3 h. The reaction is poured into ethyl acetate (50 mL)and washed with water, brine, dried over magnesium sulfate filtered, andconcentrated under reduced pressure. The residue is purified by silicagel column chromatography (50:50 ethyl acetate-hexane) to afford thedesired compound INT-20.

Step4:1-[(3-carbamimidamidopropanoyl)oxy]-3-[({1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl}carbamoyl)oxy]propan-2-yl3-carbamimidamidopropanoate (326)

INT-20 (0.20 mmol) is treated with 4N HCl/dioxane (2.0 mmol). Themixture is stirred at room temperature overnight and then water is addedand the mixture is stirred an additional 8 h. The reaction isconcentrated under reduced pressure and the residue is then purified byreverse phase chromatography (acetonitrile/water with 0.05%trifluoroacetic acid) and the product is collected and lyophilized toafford the trifluoroacetate salt of compound 326.

Example 2. Determining Compound Activity

The invention provides compounds that are useful for treating cancerand/or for inhibiting cancer cell survival, hypoxic survival, metastaticsurvival, or metastatic colonization.

To determine the activity of a compound, one can contact the compoundwith a system containing test cells expressing a reporter gene encodedby a nucleic acid operatively liked to a promoter of a marker geneselected from the above-mentioned metastasis promoters or suppressors.The system can be an in vitro cell line model or an in vivo animalmodel. The cells can naturally express the gene, or can be modified toexpress a recombinant nucleic acid. The recombinant nucleic acid cancontain a nucleic acid coding a reporter polypeptide to a heterologouspromoter. One then measures the expression level of the miRNA,polypeptide, or reporter polypeptide.

For the polypeptide, the expression level can be determined at eitherthe mRNA level or at the protein level. Methods of measuring mRNA levelsin a cell, a tissue sample, or a body fluid are well known in the art.To measure mRNA levels, cells can be lysed and the levels of mRNA in thelysates or in RNA purified or semi-purified from the lysates can bedetermined by, e.g., hybridization assays (using detectably labeledgene-specific DNA or RNA probes) and quantitative or semi-quantitativeRT-PCR (using appropriate gene-specific primers). Alternatively,quantitative or semi-quantitative in situ hybridization assays can becarried out using tissue sections or unlysed cell suspensions, anddetectably (e.g., fluorescent or enzyme) labeled DNA or RNA probes.Additional mRNA-quantifying methods include RNA protection assay (RPA)and SAGE. Methods of measuring protein levels in a cell or a tissuesample are also known in the art.

To determine the effectiveness of a compound to treat cancer and/orinhibiting cancer cell survival, hypoxic survival, metastatic survival,or metastatic colonization, one can compare the level obtained in themanner described above with a control level (e.g., one obtained in theabsence of the candidate compound). The compound is identified as beingeffective if (i) a metastasis suppressor's level is higher than acontrol or reference value or (ii) a metastasis promoter's level islower than the control or reference value. One can further verify theefficacy of a compound thus-identified using the in vitro cell culturemodel or an in vivo animal model as disclosed in the examples below.

The activity of compounds may also be determined by methods known in theart to determine CKB or creatine transport inhibition. For example,methods to determine inhibition of CKB are described in McLaughlin etal. J. Biol. Chem. 1972, 247:4382-4388, incorporated herein byreference. Methods to determine inhibition of creatine transport aredescribed in Fitch et al. Metabolism, 1980, 29:686-690, Dodd et al. J.Biol. Chem. 2005, 280:32649-32654, and Dodd et al. J. Biol. Chem. 2007,282:15528-15533, each of which is incorporated herein by reference.

Example 3. In Vivo Selection

As a first step to identify molecular regulators of liver colonizationby colon cancer, an in vivo selection was performed on the LS-174T humancolon cancer line for enhanced liver colonization through iterativeintra-hepatic injection of cancer cells into immunodeficient micefollowed by surgical resection of the liver colonies and dissociation ofcells. More specifically, liver colonization by 5×10⁵ LS-Parental, LvM3aand LvM3b cells was examined after direct intrahepatic injection bybioluminescence. Mice were imaged at day 21 after injection and liversextracted for ex vivo imaging and gross morphological examination.Photon flux ratios for the groups were obtained and compared. It wasfound that third-generation liver colonizers LS-LvM3a and LS-LvM3bdisplayed dramatically enhanced (>50 fold) capacity for livercolonization upon intra-hepatic injection relative to their parentalline. Importantly, these derivatives also displayed significantlyenhanced (>150 fold) liver metastatic capacity upon portal circulationinjection in metastatic colonization assays—revealing liver colonizationcapacity to be a key step in colon cancer metastatic progression. Forthese bioluminescence assays, all P values for the groups' respectivephoton flux ratios were based on one-sided Student's t-tests and foundto be less than 0.05, 0.001, or 0.0001.

In order to systematically identify microRNA regulators of metastaticprogression, a library of lentiviral particles, each encoding one of 611human microRNAs, was transduced into the LS-LvM3b colonizer population,the LS-174T parental line, as well as the SW620 colon cancer population.These cancer populations, containing cancer cells expressing each of 661miRNAs, were then intra-hepatically injected into mice in order to allowfor the selection of cells capable of colonizing the liver. Genomic PCRamplification of miRNA sequences, reverse-transcription, and miRNAprofiling of miRNA inserts allowed for the quantification of miRNAinsert representation. It was identified that several miRNAs displayeddrop-out in the context of liver colonization in both colon cancer celllines, consistent with the over-expression of these miRNAs suppressingliver colonization by colon cancer cells.

Example 4. Determination of Effect of Endogenous Levels of miRNAs

In this example, assays were carried out to examine whether endogenouslevels of any of these miRNAs exhibit silencing in highly metastaticderivatives relative to isogenic poorly metastatic cells. Indeed,miR-483-5p and miR-551a were found to be silenced in highly metastaticLS-LVM3a and LS-LVM3b liver colonizers relative to their parental lineand the metastatic SW620 derivative relative to its isogenic parentalline. Consistent with a suppressive role for these miRNAs in livercolonization, over-expression of miR-483-5p or miR-551a robustlysuppressed metastatic colonization by the LS-LvM3b cells, whileinhibition of endogenous miR-483-5p or miR-551a in poorly metastaticparental lines LS-174T and SW480 significantly enhanced liver metastaticcolonization.

Example 5. Investigation of the Mechanism of Action of miRNAs

In this example, assays were carried out to investigate the mechanism(s)by which these miRNAs exert their anti-metastatic effects. The effectsof these miRNAs on metastatic progression were not secondary tomodulation of proliferative capacity since miR-551a inhibition did noteffect in vitro proliferation, while miR-483-5p inhibition increasedproliferation. Additionally, over-expression of these miRNAs did notalter the invasive capacity or apoptotic rates of cancer cells. In orderto determine the mechanism(s) by which these miRNAs impact metastasis,assays were performed to identify the time-point during the metastaticprocess when cells over-expressing these miRNAs display a defect inprogression. Surprisingly, it was noted that as early as 24 hours afterinjection of cells into the portal circulation for hepatic metastaticcolonization assays, cells over-expressing these miRNAs wereout-competed in their representation relative to cells expressing acontrol hairpin.

Example 6. Organotypic Slice Culture System

To elucidate the mechanism(s) by which these miRNAs suppress livermetastatic colonization, an in vitro liver organotypic slice culturesystem was developed. This system allowed one to study early eventsduring liver metastasis after single-cell dissemination of colon cancercells in the liver microenvironment. Consistent with prior studies on asignificant selection on cell survival during metastatic colonization,there was a large drop-off in the numbers of cells within the livermicroenvironment as a function of time. Highly metastatic LvM3bcolonizer cells were significantly better at persisting in the livermicroenvironment than their poorly metastatic parental line—consistentwith a positive role for intrahepatic persistence in metastaticprogression.

Next, assays were carried out to investigate whether the effects of thismiRNA regulatory network on cancer cell persistence are caused bydiminished cancer cell survival during metastatic progression. Toquantify cell death in vivo, a bioluminescence-based luciferin reporterof caspace-3/7 activity was utilized.

More specifically, SW480 cells whose endogenous miR-483-5p or miR-551awere inhibited and subsequently introduced into the liver ofimmunodeficient mice by intrasplenic injection. Then, relative in vivocaspase activity in these cells was monitored using a caspase-3activated DEVD-luciferin. It was found that miRNA inhibitionsignificantly reduced in vivo caspase activity in colon cancer cellsduring the early phase of hepatic colonization, revealing cancersurvival to be the phenotype suppressed by these miRNAs.

These in vivo findings were corroborated by an organotypic slice culturesystem. Briefly, survival of the SW480 cells in organotypic cultures(n=8) whose endogenous miR-483-5p or miR-551a were inhibited bypre-treatment with LNAs. 5×10⁵ cells were labeled with cell-trackergreen (LS-Parental) or cell-tracker red (LvM3b) and introduced into theliver through intrasplenic injection. Immediately after injection, theliver was excised and 150-um slice cultures were made using a tissuechopper. Survival of the cells in organotypic cultures was monitored forup to 4 days with a multi-photon microscope. Dye-swap experiments wereperformed to exclude effects of dye bias. Representative images at day 0and day 3 were shown. It was found that over-expression of bothmicroRNAs in LS-LvM3b cells suppressed colon cancer persistence whileinhibition of endogenous levels of both microRNAs enhanced persistenceof poorly metastatic SW480 cells. The above findings reveal miR-483-5pand miR-551a to suppress liver metastatic colonization and metastaticcell survival in the liver—a phenotype exhibited by highly metastaticcolon cancer cells.

Example 7. Investigation of Downstream Effectors

In this example, assays were carried out to identify the downstreameffectors of these miRNAs. Through transcriptomic profiling, transcriptsthat were down-regulated by over-expression of each microRNA and whichcontained 3′-UTR or coding-sequence (CDS) elements complementary to themiRNAs were identified. Interestingly, Creatine Kinase Brain-type (CKB)was identified as a putative target of both miRNAs, suggesting thatthese miRNAs, which exhibit common in vivo and organotypic phenotypesmight mediate their effects through a common target gene. Indeed,quantitative PCR validation revealed suppression of CKB transcriptlevels upon over-expression of the microRNAs. It was found thatexpression levels of CKB in highly metastatic LvM3b cells weresuppressed by over-expressing miR-483-5p and miR-551a. Additionally,endogenous miR-483 and miR-551a were found to suppress endogenous CKBprotein levels. For example, it was found that expression of CKB wasup-regulated in poorly metastatic SW480 cells whose endogenousmiR-483-5p and miR-551 aa were inhibited with LNAs. Mutagenesis andluciferase-based reporter assays revealed miR-483-5p and miR-551a todirectly target the 3′UTR or CDS of CKB. To that end, luciferasereporter assays of CKB coding sequence and 3′-UTR were carried out. Itwas found that miR-483-5p and miR-551a targeted complementary regions inthe 3′-UTR and coding sequence of CKB respectively. The assays wereperformed with the complementary regions mutated as well and they wereperformed at least 3 times.

Example 8. Investigation of the Role of CKB in Liver Metastasis

In this example, assays were carried out to examine if CKB is sufficientand necessary for liver metastatic colonization by colon cancer.

Briefly, liver metastasis was examined in mice injected intrasplenicallywith 5×10⁵ poorly metastatic SW480 cells and CKB over-expressing cells.The mice were euthanized at 28 days after injection and livers excisedfor bioluminescent imaging. Similarly, liver metastasis was alsoexamined in mice injected intrasplenically with 5×10⁵ highly aggressiveLvM3b expressing a control hairpin or a hairpin targeting CKB. Thesemice were euthanized 21 days after injection as described above.

It was found that over-expression of CKB in poorly metastatic SW480cells was sufficient to promote liver metastasis by more than 3-folds,while CKB knockdown in metastatic LS-LvM3b cells and SW480 cells,through independent hairpin knockdown in each line robustly suppressedliver metastasis by more than 5 folds. Consistent with the effects ofthe miRNAs, CKB over-expression was sufficient to significantly enhancethe ability of colon cancer cells to persist in the livermicro-environment and enhanced their representation in the liver, whileCKB knockdown significantly reduced intra-hepatic persistence. To thatend, study was carried out to examine survival of control SW480 and CKBover-expressing SW480 cells in organotypic liver slices (n=8), andorganotypic slice cultures of LvM3b cells expressing a control hairpinor hairpin targeting CKB (n=8). Images taken at day 0 and day 2 showedthat CKB over-expression was sufficient to significantly enhance theability of cancer cells. In these assays, P values were found to be lessthan 0.001 or 0.0001 based on one-sided Student's t-tests.

To investigate whether CKB acts directly downstream of miR-483-5p andmiR-551a, the coding-sequence of CKB was over-expressed in cellsover-expressing miR-483-5p or miR-551a. Briefly, assays were performedto examine metastatic progression in mice injected with 5×10⁵ LvM3bcells over-expressing miR-483-5p and miR-551a, with and without CKBover-expression. Liver metastases were monitored by bioluminescentimaging and mice euthanized 35 days after injection. It was found thatover-expression of CKB was sufficient to rescue the suppressed livermetastatic phenotypes of cells over-expressing miR-483-5p and miR-551a.Conversely, knockdown of CKB in cells displaying endogenous miR-483-5por miR-551a inhibition prevented the enhanced metastasis effect seenwith miR-483-5p or miR-551a inhibition. To that end, assays wereperformed to examine liver metastasis in mice injected with 5×10⁵ SW480cells whose endogenous miR-483-5p and miR-551a were inhibited by LNA,with and without CKB knockdown. The mice were euthanized after 28 daysand liver excised for ex vivo bioluminescence imaging. The results ofthe above mutational, gain- and loss-of-function experiments, andepistasis analyses reveal CKB to be a direct target of miR-483-5p andmiR-551a and to act as a downstream effector of these miRNAs in theregulation of colon cancer metastatic progression. In these assays, Pvalues were found to be less than 0.05 or 0.001 based on one-sidedStudent's t-tests.

To further confirm the roles of CKB, relative in vivo caspase activitieswere examined in control SW480 and CKB over-expressing cells in liversof mice. The activities were measured by bioluminescence using acaspase-3 activated DEVD-luciferin and normalized to bioluminescencesignal from regular luciferin (n=3). Similar relative in vivo caspase-3activity were also examined in SW480 cells expressing a control hairpinor hairpin targeting CKB and introduced into the livers of mice throughintrasplenic injection. Caspase activities were measured on day 1, day 4and day 7 after injection. Consistent with the above findings, CKBover-expression significantly reduced, while CKB knockdown significantlyenhanced, in vivo caspase-3/7 activity in colon cancer cells during theinitial phase of hepatic colonization. In these assays, P values werefound to be less than 0.05 or 0.001 based on one-sided Student'st-tests. These findings reveal CKB to be a promoter of colon cancersurvival during hepatic metastatic colonization.

Example 9. CKB Knockdown Experiments

CKB is known to regulate the reservoir of rapidly mobilized high-energyphosphates in tissues such as the brain and kidneys by catalyzing thetransfer of a high-energy phosphate group from phosphocreatine to ADP,yielding ATP and creatine. It was hypothesized that CKB generation ofATP from phosphocreatine might provide colon cancer cells with anenergetic advantage during hepatic colonization. To determine if ATP,the end-product of CKB catalysis, could rescue metastasis suppressionseen upon CKB knockdown, CKB knockdown cells were loaded with ATP priorto injection of cells in experimental metastasis assays. Briefly, livermetastasis was examined in mice injected with 5×10⁵ LvM3b with orwithout CKB knockdown and pre-treated with 100 uM ATP or vehicle.Metastatic burden was monitored by bioluminescent imaging and miceeuthanized 21 days after injection. It was found that ATP loading ofcells was sufficient to significantly enhance the suppressed metastasisphenotype in cells depleted of CKB by more than 10 folds. The rescue byATP was specific since ATP loading did not enhance the metastaticactivity of cells expressing a short-hairpin control.

Similar studies were done to determine whether creatine andphosphocreatine could rescue the phenotype of seen upon CKB knock-down.More specifically, assays were performed to examine liver metastasis inmice injected with 5×10⁵ LvM3b cells pre-treated with 10 uM creatine, inthe background of CKB knockdown. The mice were then euthanized asdescribed above and liver extracted for ex vivo bioluminescent imagingat day 21 after injection. Also, colorectal cancer metastasis wasexamined in mice injected with 5×10⁵ LvM3b cells with CKB knockdown andpre-treated with 10 uM creatine-phosphate. Liver metastasis wasmonitored by bioluminescent imaging and mice were euthanized asdescribed above. It was found that both creatine and creatine-phosphaterescued metastasis suppression.

In order to investigate whether colon cancer metastasis could beinhibited by blocking the transport of creatine into colon cancer cells,the creatine transporter channel SLC6a8 was inhibited in LvM3b cells byexpressing short hairpin targeting SLC6a8. Then liver metastasis byLvM3b cells was examined in the same manner described above. It wasfound that knock-down of the creatine transporter channel SLC6a8inhibited colon cancer metastasis. These findings reveal that coloncancer cells are dependent on CKB generated ATP for their survivalduring hepatic colonization.

Example 10. Analysis of miRNA and CKB Expression Levels in Primary ColonCancers and Liver Metastases

In order to determine if this cooperative miRNA regulatory networkcontrolling colon cancer metastatic progression has human pathologicrelevance, the expression levels of miR-483-5p and miR-551a wereanalyzed in a set of 67 primary colon cancers as well as livermetastases obtained from patients at MSKCC. More specifically,miR-483-5p and miR-551a levels in 37 primary tumor samples and 30 livermetastases samples were quantified by quantitative real-time PCR.Consistent with a metastasis-suppressive role for these miRNAs duringmetastatic progression, miR-483 and miR-551a both displayedsignificantly reduced expression levels in human liver metastasesrelative to primary colon cancers (FIG. 1a ; p<0.05 for miR-483-5p andp<0.05 for miR-551a; N=67).

CKB expression levels were also examined in the 37 primary tumor samplesand 30 liver metastases samples by quantitative real-time PCR.Importantly, CKB expression was found to be significantly elevated inliver metastases relative to primary colon cancers (p<0.05) and itsexpression was significantly anti-correlated with the miRNAs—consistentwith its direct targeting by these miRNAs in human colon cancer (FIG. 1b). These findings are consistent with previous clinical histologicanalyses revealing elevated levels of CKB protein in advanced stagecancer.

Example 11. Investigation of miRNA Regulatory Network as a TherapeuticTarget

In this example, assays were carried out to investigate the therapeuticpotential of targeting this miRNA regulatory network. To this end, micewere injected with a high number (500k) of highly metastatic LvM3a cellsand 24 hours later injected mice with a single intra-venous dose ofadenoviral-associated virus (AAV) expressing miR-483-5p and miR-551a offa single transcript. It was found that a single therapeutic dose ofadeno-associated virus (AAV) delivering both miRNAs dramatically andsignificantly reduced metastatic colonization by more than 5 fold (FIG.1c ).

Finally, assays were carried out to determine the impact ofsmall-molecule inhibition of CKB and restriction of creatineavailability on colon cancer metastasis. Cyclocreatine, which resemblesphosphocreatine, is a transition-state analog for creatine kinases. Toexamine the effect of cyclocreatine, bioluminescent measurements ofliver metastasis were carried out in mice injected with 5×10⁵ LvM3bcells and treated with cyclocreatine daily for two weeks. The mice werethen euthanized and livers excised for ex vivo imaging at the end of thetreatment. It was found that, despite being a poor inhibitor of CKB(5000 uM ki), treatment of mice with cyclocreatine significantly reducedmetastatic colonization and proved superior to the currentstandard-of-care FOLFOX chemotherapy (FIG. 1d ).

Similar assays were carried out using a creatine transporter inhibitorbeta-guanidinopropionic acid (β-GPA). Bioluminescent measurements wereused to examine liver metastasis in mice injected with 5×10⁵ LvM3b cellsand treated with β-GPA daily for two weeks. It was found that treatmentof mice with this competitive inhibitor of the creatine transporterchannel also significantly reduced metastatic colonization (FIG. 1e ).

Using a systematic approach, two miRNAs were identified to act assuppressors of liver metastatic colonization by colon cancer cells. Itwas found find that these miRNAs convergently target CKB—a key gene thatendows cells encountering hepatic hypoxia with the ability to generateATP from phosphocreatine reserves. The successful targeting of thispathway using 4 independent therapeutics that were more effective thanthe current clinical standard-of-care, and which displayed no apparenttoxicity suggest promise for therapeutic targeting of this pathway inhuman colon cancer. The above-described combined in vivo selection/genescreening approach, which is designated as MUlti-Gene Screening of Humangenes through intra-Organ Tandem Selection (MUGSHOTS) has efficientlyidentified robust and pathologically validated regulators of livercolonization and metastasis by colon cancer and has the potential todiscover coding and non-coding regulators of metastatic colonization ofany organ by any cancer type.

Example 12. Investigation of Creatine Transport as a Therapeutic Target

In this example, assays were carried out to confirm the therapeuticpotential of targeting the creatine transporter channel SLC6a8 byadministering the small molecule β-GPA, which is an inhibitor of SLC6a8.As mentioned above, administration of β-GPA to mice injected with LvM3bcolon cancer cells resulted in inhibition of colon cancer metastasis tothe liver after two weeks of treatment (FIG. 1e ). To confirm thistherapeutic effect, mice injected with LvM3b colon cancer cells wetreated with either 6-GPA or control vehicle (PBS) via intra-peritonealinjection daily for three weeks (FIG. 2). The mice were euthanized atthree weeks and liver extracted for bioluminescent imaging and grosshistology.

It was found that daily treatment with β-GPA led to a significantreduction in colon cancer metastasis to the liver, as assessed by invivo bioluminescent imaging of in vivo mice, bioluminescent imaging ofextracted liver, and by gross anatomical examination of extracted liversfrom treated mice (FIG. 2). More specifically, the average photon fluxratios as measure by the bioluminescence imaging for the control group(without treatment of β-GPA) and the treated groups were about 800 and100, respectively. P values were found to be less than 0.05 based onone-sided Student's t-tests.

Example 13. Knockdown of SLC6a8

In this example assays were carried out to evaluate the therapeuticbenefit of targeting the creatine transporter channel SLC6a8 with shRNAknockdown targeting SLC6a8.

Briefly, mice were injected with LvM3b colon cancer cells expressingeither of two independent short hairpin RNAs (shSLC6a8 #4 or shSLC6a8#5) targeting the creatine transporter channel SLC6a8 or with controlRNA (empty pLKO vector, ordered from Sigma Aldrich) (FIG. 3a ). Again,liver metastasis was monitored by bioluminescent imaging and mice wereeuthanized three weeks after inoculation of cancer cells. Livers wereextracted for gross histology. It was found that knockdown of SLC6a8with two independent shRNAs resulted in inhibition of colon cancermetastasis (FIG. 3a ).

To further confirm the therapeutic benefit of knockdown of SLC6a8,another independent colon cancer cell line (SW480 colon cancer cellline) expressing a short hairpin RNA targeting SLC6a8 (shSLC6a8 #2) wasinjected into mice (FIG. 3b ). It was found that SLC6a8 knockdownsignificantly inhibited metastasis of SW480 colon cancer cells (FIG. 3b).

Lastly, the therapeutic benefit of targeting SLC6a8 was investigated inpancreatic cancer cells. To accomplish this, PANC1 pancreatic cancercells expressing either an shRNA targeting SLC6a8 (shSLC6a8 #5) or acontrol RNA (empty pLKO vector) were injected into mice. Metastaticprogression was monitored by bioluminescent imaging and mice wereeuthanized in the same manner described above. It was found that, at 28days, there was a significant reduction in pancreatic cancer metastasisin the cells treated with shRNA targeting SLC6a8, revealing that SLC6a8is a therapeutic target for pancreatic cancer.

Example 14. Correlation of Creatine Transport and Metastatic Progression

In this example, it was investigated whether expression of the creatinetransporter SLC6a8 in human colon cancer tumors correlated withmetastatic progression.

To accomplish this, quantitative real-time PCR was used to quantify theexpression of SLC6a8 in 36 primary colon cancer tumors and 30 metastaticcolon cancer tumors (FIG. 4). Indeed, expression of SLC6a8 wassignificantly higher in metastatic tumors (about 1.3) as compared withprimary tumors (about 0.5), further confirming the central role ofSLC6a8 in metastasis (FIG. 4). P values were found to be less than 0.05based on one-sided Student's t-tests.

Example 15. Effect of β-GPA on Pancreatic Cancer

As mentioned above, it was demonstrated that inhibition of the creatinetransporter SLC6a8 with shRNA mediated knock-down resulted insuppression of metastasis of both colon cancer as well as pancreaticcancer. It was also demonstrated that inhibition of SLC6a8 with thesmall molecule inhibitor β-GPA resulted in therapeutic benefit for coloncancer metastasis in vivo. To evaluate if β-GPA treatment results intherapeutic benefit in pancreatic cancer, the ability of β-GPA treatmentto inhibit the survival of human pancreatic cancer cells was assessed invivo in mice.

Briefly, PANC1 pancreatic cancer cells were incubated for 48 hours withand without the presence of 10 mM of β-GPA, then injected intoimmunodeficient mice (5×10⁵ PANC1 cells each mouse; 4 mice each in thetreated and untreated cohort). The mice were imaged with bioluminescenceimaging on day 1 after injection and signal was normalized to day zero.Therapeutic benefit was observed as early as one day after theinjections, with a significant reduction in the tumor burden ofpancreatic cancer cells in vivo as assessed by bioluminescence imaging(FIG. 4) demonstrating therapeutic benefit of β-GPA treatment forpancreatic cancer. More specifically, the average photon flux ratios asmeasure by the bioluminescence imaging for the control group (withouttreatment of β-GPA) and the treated groups were about 2.7 and 1.6,respectively. P values were found to be less than 0.05 based onone-sided Student's t-tests.

Example 16. Combination of β-GPA and Fluorouracil or Gemcitabine

The above examples demonstrated that β-GPA treatment alone resulted intherapeutic benefit for colon cancer and pancreatic cancer. In thisexample it was investigated whether β-GPA treatment could enhance thetherapeutic activity of the chemotherapy agents 5′-Fluorouracil andGemcitabine. To accomplish this, cell viability was performed assays tocompare the cytotoxic activity of 5′-Fluorouracil or Gemcitabine alonecompared with combined therapy with β-GPA.

Briefly, 10 000 PANC1 cells were seeded in triplicate in 96-well platesand treated with various concentrations of Gemcitabine (1 nm, 10 nm, 100nm, 1000 nm, 10000 nm, 100000 nm, and 1000000 nm) with or without 10 mMof β-GPA for 48 hours. Cell viability was then assayed using the WST-1reagent (Roche Applied Science), with absorbance at 440 nm an indicatorof the number of viable cells. As shown in FIG. 6, it was found that theaddition of a therapeutic concentration of β-GPA enhanced the cytotoxicactivity of Gemcitabine on PANC1 pancreatic cancer cells as assessed bya cell viability assay using the WST-1 reagent.

Likewise, the addition of a therapeutic concentration of β-GPA enhancedthe cytotoxic activity of 5′-Fluorouracil on Ls-LvM3b colon cancercells. To that end, 10,000 Ls-LvM3b cells were seeded in triplicate in96-well plates and treated with various concentrations of5′-Fluorouracil with or without 10 mM of β-GPA for 48 hours. Cellviability was assayed in the same manner described above with absorbanceat 440 nm an indicator of the number of viable cells. As shown in FIG.7, these results demonstrate that β-GPA enhance the therapeutic activityof commonly utilized chemotherapeutic agents for the treatment ofcolorectal and pancreatic cancer.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated herein in their entireties.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features herein before set forth.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A compound having the structure:

wherein Q1 is

m is 1 or 2; R7 is hydrogen; one R8 combines with R12 with the atoms towhich they are attached to form an optionally substituted C3-C4heterocycle; a second R8, if present, is hydrogen, deuterium, halo, NH2,optionally substituted C1-C3 alkyl, or combines with an R9 and with theatoms to which they are attached to form an optionally substituted C3-C6cycloalkyl ring; or combines with R10 or R11 and with the atoms to whichthey are attached to form an optionally substituted C3-C4 cycloalkylring; each R9 is independently hydrogen, deuterium, halo, NH2,optionally substituted C1-C3 alkyl or combines with the second R8, ifpresent, and with the atoms to which they are attached to form anoptionally substituted C3-C6 cycloalkyl ring; or combines with R10 orR11 and with the atoms to which they are attached to form an optionallysubstituted C3-C4 cycloalkyl ring; R10 and R11 are independentlyhydrogen, deuterium, optionally substituted C1-C4 alkyl, optionallysubstituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl or R10and R11 combine with the atoms to which they are attached to form anoptionally substituted C3-C6 cycloalkyl ring; or R10 or R11 combine withR8 or R9 with the atoms to which they are attached to form an optionallysubstituted C3-C4 cycloalkyl ring; or R10 or R11 combine with R12 withthe atoms to which they are attached to form an optionally substitutedC3-C4 heterocycle; R12 is hydrogen, optionally substituted C1-C6 alkyl,or R12 combines with R8 or R9 with the atoms to which they are attachedto form an optionally substituted C3-C4 heterocycle, or R12 combineswith R10 or R11 with the atoms to which they are attached to form anoptionally substituted C3-C4 heterocycle; wherein if m is 1 and R10 ismethyl then at least one of R9, and R11 is not hydrogen; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein R¹⁰ is optionally substituted C₁-C₄ alkyl, optionallysubstituted C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl.
 3. Thecompound of claim 2, wherein R¹⁰ is methyl, ethyl, n-propyl, iso-propyl,—CD₃, —CF₃, —CH₂F, —CHF₂, —CH═CH₂, or —C≡CH.
 4. The compound of claim 2,wherein R¹¹ is hydrogen or methyl.
 5. The compound of claim 2, wherein mis 2 and the second R⁸ is hydrogen.
 6. The compound of claim 2, whereinR⁹ is hydrogen, NH₂, or methyl.
 7. The compound of claim 1, wherein saidoptionally substituted C3-C4 heterocycle is an optionally substituted C4heterocycle.
 8. A compound having the structure of Formula VIII:

wherein b is 0 or 1 and c is 0; Q1 is

R9 is hydrogen, deuterium, halo, NH2, optionally substituted C1-C3alkyl; and R10 and R11 are, independently, hydrogen, deuterium,optionally substituted C1-C4 alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6 alkynyl, or a pharmaceuticallyacceptable salt thereof.
 9. The compound of claim 1, wherein R10 ishydrogen or optionally substituted C1-C4 alkyl.
 10. The compound ofclaim 9, wherein R10 is hydrogen or methyl.
 11. The compound of claim 1,wherein R11 is hydrogen.
 12. The compound of claim 1, wherein R9 ishydrogen, halo, hydroxyl, NH2, optionally substituted C1-C3 alkyl. 13.The compound of claim 12, wherein R9 is hydrogen, fluoro, hydroxyl, NH2,or methyl.
 14. A compound having the structure:

or a pharmaceutically acceptable salt thereof.
 15. A compound having thestructure:

or a pharmaceutically acceptable salt thereof.
 16. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable excipient.